Search

BackgroundEnterovirus (EV) and parechovirus type A3 (PeV-A3) cause infections ranging from asymptomatic to life-threatening. Host immune responses in children, particularly innate responses to PeV-A3, remain largely unknown. The aim of this study was to determine aspects of the cytokine/chemokine responses to EV and PeV-A3 in cerebrospinal fluid (CSF) and plasma obtained from children with systemic/central nervous system infection.MethodsA total of 74 salvaged CSF samples (27 with EV, 23 with PeV-A3, and 24 with neither EV nor PeV-A3) and 35 paired blood samples (10 with EV, 14 with PeV-A3, and 11 with neither) were studied. Concentrations of cytokines and chemokines were measured using a customized 21-plex MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel. Additionally, clinical characteristics data for all the patients were collected from electronic medical records to evaluate the potential association between the immune response and presentations.ResultsWe demonstrate that EV and PeV-A3 infections induce different cytokine/chemokine immune responses in children. EV induces more robust responses in CSF with significantly elevated levels of fractalkine, interferon (IFN)-α2, IFN-γ, interleukin (IL)-1Rα, IL-4, IL-8, and tumor necrosis factor α; PeV-A3 induces less robust or absent responses in CSF but robust responses in plasma, with significantly higher concentrations of IFN-α2, IL-15, IL-1Rα, interferon-γ-inducible protein–10, and monocyte chemoattractant protein–1.ConclusionsHigh cytokine/chemokine concentrations in the plasma of PeV-A3 patients compared with EV patients could explain higher/more prolonged fever in PeV-A3 patients, whereas relatively low cytokine/chemokine concentrations in PeV-A3 CSF might explain the absence of CSF pleocytosis.

Highlights • Changing our respiratory panel assay decreased turn-around-time from 27.1 to 1.4 h. • The faster assay was associated with decreased empiric antibiotic use, less x-rays, and shorter LOS. • The faster assay improved optimal use of oseltamivir in influenza infected patients., Background Empiric antibiotic treatment is common among children with acute respiratory tract infections (ARTI), despite infections being predominately viral. The use of molecular respiratory panel assays has become increasingly common for medical care of patients with ARTIs. Study design This was a 6-year retrospective, single-centered study of pediatric inpatients who tested positive for an ARTI respiratory pathogen. We examined the relationship between clinical outcomes and whether the patient was tested using the Luminex Respiratory Viral Panel ([RVP]; in-use: Dec. 2009 – Jul. 2012) or Biofire Respiratory Pathogen Panel ([RP]; in-use Aug. 2012 – Jun. 2016). The prevalence and duration of pre-test empiric antibiotics, post-test oseltamivir administration to influenza patients, chest x-rays and length of stay between the two assays was compared. Results A total of 5142 patients (1264 RVP; 3878 RP) were included. The median laboratory turn-around-time for RP was significantly shorter than RVP (1.4 vs. 27.1 h, respectively; p

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 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

Background Urinalysis (UA) has been routinely used as a screening tool prior to microbial culture set-up in many laboratories. BacterioScan 216Dx instrument utilizes laser light scattering technology to detect bacterial growth in urine and results are available in 3 hours. The aim of this study was to compare the performance of 216 DX and UA against culture as gold standard. Methods Clean-catch, unpreserved, either UA positive (leukocyte esterase > trace, or nitrite positive or white blood cells >5/hpf) or UA negative samples from children aged ≤18 years were tested by 216Dx within 24 hours of sample collection. “ Likely positive” samples by 216Dx were tested by MALDI-TOF for direct bacterial identification. Sensitivity and specificity of 216Dx and UA was determined against urine culture. Results Total of 205 urine samples were included in this study, of which 48.0% (98/205) and 52.0% (107/205) were UA positive and negative, respectively. 77.0% of samples were collected from female and median age was 108 months. Overall sensitivity, specificity, positive, and negative predictive value (PPV and NPV) of 216Dx and UA are shown in table below. Of 27 true positive (TP) samples by 216Dx, 77.0% (21/27) were successfully identified by MALDI-TOF. There were a total of 96 samples identified as contamination/normal flora by culture. Among these, 63 samples (65.0%) were detected as true negative (TN) by 216Dx vs. 50 samples (53.1%) as TN by UA. Two false negative (FN) samples by 216Dx and one FN by UA were K. oxytoca, S. epidermidis (both >100 K cfu/ml) and E. coli (>100 K cfu/mL), respectively. Conclusion Although sensitivity of both 216Dx and UA is comparable, specificity of 216Dx was higher than UA. Also, 216Dx showed better performance in detecting urine contamination, thus reducing laboratory reagent and labor cost. Faster turn-around-time of 216Dx coupled with rapid identification of uropathogen by MALDI-TOF has the potential to reduce unnecessary antibiotic use, improve patient management and reduce overall healthcare-related cost. Assay TP FP TN FN %Sensitivity (95% CI) %Specificity (95% CI) %PPV (95% CI) %NPV (95% CI) 216Dx 27 36 140 2 93.1 (75.7–98.8) 79.5 (72.6.0-85.1) 42.8 (30.6–55.9) 98.6 (94.4–99.7) UA 28 70 106 1 96.5 (80.3–99.8) 60.2 (52.5–67.4) 28.5 (20.1–38.7) 99.0 (94.1–99.9) Disclosures All authors: No reported disclosures.

Summary Background 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. Methods 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 χ 2 tests were used to test for statistical significance. Findings 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). Interpretation 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. Funding None.

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.

We have proposed the novel concept that the macrophage ubiquitin-proteasome pathway functions as a key regulator of Lipopolysaccharide (LPS)-induced inflammation signaling. These findings suggest that proteasome-associated protease subunits X, Y, and Z are replaced by LMP subunits after LPS treatment of RAW 264.7 cells. The objective here was to determine the contribution of selective LMP proteasomal subunits to LPS-induced nitric oxide (NO) and TNF-α production in primary murine macrophages. Accordingly, thioglycollate-elicited macrophages from LMP7, LMP2, LMP10 (MECL-1), and LMP7/MECL-1 double knockout mice were stimulated in vitro with LPS, and were found to generate markedly reduced NO levels compared to wild-type (WT) mice, whereas TNF-α levels responses were essentially unaltered relative to wild-type responses. The recent studies suggest that the TRIF/TRAM pathway is defective in LMP knockouts which may explain why iNOS/NO are not robustly induced in LPS-treated macrophages from knockouts. Treating these macrophages with IFN-γ and LPS, however, reverses this defect, leading to robust NO induction. TNF-α is induced by LPS in the LMP knockout macrophages because IκB and IRAK are degraded normally via the MyD88 pathway. Collectively, these findings strongly support the concept that LMP7/MECL-1 proteasomes subunits actively function to regulate LPS-induced NO production by affecting the TRIF/TRAM pathway.

Summary The effect of lipopolysaccharide (LPS) on the in vivo lethal action of doxorubicin (DOX) against mice was studied. DOX killed LPS-pretreated mice much earlier than untreated mice, and exhibited a stronger toxic action against LPS-pretreated mice. DOX-induced lethality in LPS-pretreated mice was due to severe hepatic damage, but there were no significant lesions in the heart, kidney and lung. Hepatic lesions were accompanied by caspase 3-positive cells and fragmented DNA-positive cells, suggesting the involvement of apoptosis. DOX induced the production of a high level of interferon (IFN)-γ and tumour necrosis factor (TNF)-α in LPS-pretreated mice, but not in non-treated mice. The DOX-induced lethality was prevented significantly by anti-IFN-γ antibody, but not anti-TNF-α antibody. Administration of recombinant IFN-γ in place of LPS augmented definitively the DOX-induced lethality. LPS augmented the DOX-induced lethality in TNF-α-deficient mice. Taken together, LPS was suggested to enhance DOX-induced IFN-γ production and augment the in vivo lethal action via hepatic damage.