Aaron L. Miller

Comparison of the FilmArray Respiratory Panel and Prodesse Real-Time PCR Assays for Detection of Respiratory Pathogens

ABSTRACT We compared the diagnostic performance and overall respiratory pathogen detection rate of the premarket version of the FilmArray Respiratory Panel (RP) multiplex PCR assay (Idaho Technology, Inc., Salt Lake City, UT) with those of the Food and Drug Administration (FDA)-cleared Prodesse ProFlu+, ProFAST+, ProParaflu+, Pro hMPV+, and ProAdeno+ real-time PCR assays (Gen-Probe, San Diego, CA). The assays were performed on a panel of 192 nasopharyngeal-secretion specimens collected from 81 children under 1 year of age with upper respiratory tract symptoms. To resolve discordant results and confirm pathogens detected only by the larger FilmArray panel, we performed laboratory-developed real-time PCR assays. Among viruses detectable by both commercial assays (adenovirus, human metapneumovirus, influenza A virus, influenza B virus, parainfluenza viruses 1 to 3, and respiratory syncytial virus), the FilmArray and Prodesse assays showed good overall agreement (181/192 [94.3%]; kappa = 0.87; 95% CI, 0.79 to 0.94). FilmArray RP detected more parainfluenza viruses 1 and 3 than ProParaflu+ (18 versus 13) while ProAdeno+ detected more adenoviruses (11 versus 6), but these differences were not statistically significant. Additionally, FilmArray RP detected 138 pathogens (confirmed as true positives) not included in the Prodesse assays (rhinovirus [RV]/enterovirus [EV], 118; bocavirus, 8; coronavirus, 7; parainfluenza virus 4, 4; Mycoplasma pneumoniae, 1). FilmArray RP was cleared by the FDA following the completion of this study. The FDA-cleared version includes the following targets: adenovirus, coronaviruses HKU1 and NL63, human metapneumovirus (hMPV), influenza A virus (to type level only), influenza A H1 seasonal virus, influenza A H3 seasonal virus, influenza A virus H1-2009, influenza B virus, parainfluenza viruses 1 to 4, respiratory syncytial virus (RSV), and RV/EV (no differentiation). The larger panel in the FilmArray RP assay allowed the detection of additional respiratory pathogens compared to the Prodesse assays. In this population of young children with upper respiratory tract infection, RV/EV accounted for the majority of the additional pathogens detected by FilmArray RP.

Viral-Bacterial Interactions and Risk of Acute Otitis Media Complicating Upper Respiratory Tract Infection

ABSTRACT Acute otitis media (AOM) is a common complication of upper respiratory tract infection whose pathogenesis involves both viruses and bacteria. We examined risks of acute otitis media associated with specific combinations of respiratory viruses and acute otitis media bacterial pathogens. Data were from a prospective study of children ages 6 to 36 months and included viral and bacterial culture and quantitative PCR for respiratory syncytial virus (RSV), human bocavirus, and human metapneumovirus. Repeated-measure logistic regression was used to assess the relationship between specific viruses, bacteria, and the risk of acute otitis media complicating upper respiratory tract infection. In unadjusted analyses of data from 194 children, adenovirus, bocavirus, Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis were significantly associated with AOM (P < 0.05 by χ2 test). Children with high respiratory syncytial virus loads (≥3.16 × 107 copies/ml) experienced increased acute otitis media risk. Higher viral loads of bocavirus and metapneumovirus were not significantly associated with acute otitis media. In adjusted models controlling for the presence of key viruses, bacteria, and acute otitis media risk factors, acute otitis media risk was independently associated with high RSV viral load with Streptococcus pneumoniae (odds ratio [OR], 4.40; 95% confidence interval [CI], 1.90 and 10.19) and Haemophilus influenzae (OR, 2.04; 95% CI, 1.38 and 3.02). The risk was higher for the presence of bocavirus and H. influenzae together (OR, 3.61; 95% CI, 1.90 and 6.86). Acute otitis media risk differs by the specific viruses and bacteria involved. Acute otitis media prevention efforts should consider methods for reducing infections caused by respiratory syncytial virus, bocavirus, and adenovirus in addition to acute otitis media bacterial pathogens.

Gene expression profile of human lymphoid CEM cells sensitive and resistant to glucocorticoid-evoked apoptosis

Three closely related clones of leukemic lymphoid CEM cells were compared for their gene expression responses to the glucocorticoid dexamethasone (Dex). All three contained receptors for Dex, but only two responded by undergoing apoptosis. After a time of exposure to Dex that ended late in the interval preceding onset of apoptosis, gene microarray analyses were carried out. The results indicate that the expression of a limited, distinctive set of genes was altered in the two apoptosis-prone clones, not in the resistant clone. That clone showed altered expression of different sets of genes, suggesting that a molecular switch converted patterns of gene expression between the two phenotypes: apoptosis-prone and apoptosis-resistant. The results are consistent with the hypothesis that altered expression of a distinctive network of genes after glucocorticoid administration ultimately triggers apoptosis of leukemic lymphoid cells. The altered genes identified provide new foci for study of their role in cell death.

Pathway interactions between MAPKs, mTOR, PKA, and the glucocorticoid receptor in lymphoid cells

BackgroundGlucocorticoids are frequently used as a primary chemotherapeutic agent in many types of human lymphoid malignancies because they induce apoptosis through activation of the glucocorticoid receptor, with subsequent alteration of a complex network of cellular mechanisms. Despite clinical usage for over fifty years, the complete mechanism responsible for glucocorticoid-related apoptosis or resistance remains elusive. The mitogen-activated protein kinase pathway is a signal transduction network that influences a variety of cellular responses through phosphorylation of specific target substrates, including the glucocorticoid receptor. In this study we have evaluated the pharmaceutical scenarios which converge on the mitogen-activated protein kinase pathway to alter glucocorticoid sensitivity in clones of human acute lymphoblastic CEM cells sensitive and refractory to apoptosis in response to the synthetic glucocorticoid dexamethasone.ResultsThe glucocorticoid-resistant clone CEM-C1-15 displays a combination of high constitutive JNK activity and dexamethasone-induced ERK activity with a weak induction of p38 upon glucocorticoid treatment. The cells become sensitive to glucocorticoid-evoked apoptosis after: (1) inhibition of JNK and ERK activity, (2) stimulation of the cAMP/PKA pathway with forskolin, or (3) inhibition of mTOR with rapamycin. Treatments 1–3 in combination with dexamethasone alter the intracellular balance of phospho-MAPKs by lowering JNK phosphorylation and increasing the level of glucocorticoid receptor phosphorylated at serine 211, a modification known to enhance receptor activity.ConclusionOur data support the hypothesis that mitogen-activated protein kinases influence the ability of certain malignant lymphoid cells to undergo apoptosis when treated with glucocorticoid. Activated/phosphorylated JNK and ERK appear to counteract corticoid-dependent apoptosis. Inhibiting these MAPKs restores corticoid sensitivity to a resistant clone of CEM cells. Forskolin, which activates the cAMP pathway, and rapamycin, which inhibits mTOR, also inhibit JNK. Further, the sensitizing treatments result in a largely dexamethasone-dependent increase in the total pool of glucocorticoid receptor phosphorylated at serine 211. The phospho-serine 211 receptor is known to be more potent in activating gene transcription and apoptosis. The interactive effects demonstrated here in reverting resistant cells to corticoid sensitivity could provide therapeutic clinical potential in the treatment of lymphoid malignancies.

Symptomatic and Asymptomatic Respiratory Viral Infections in the First Year of Life: Association With Acute Otitis Media Development

Sensitive viral diagnostic methods have identified increasing prevalence of asymptomatic viral infection. This study determined the epidemiologic characteristics and etiology of asymptomatic upper respiratory tract infection in the first year of life and the association with acute otitis media complication.

Gene networks in glucocorticoid-evoked apoptosis of leukemic cells.

To discover the genes responsible for the apoptosis evoked by glucocorticoids in leukemic lymphoid cells, we have begun gene array analysis on microchips. Three clones of CEM cells were compared: C7-14, C1-15 and C1-6. C7-14 and C1-15 are subclones from the original clones C7 (sensitive to apoptosis by glucocorticoids) and C1 (resistant). C1-6 is a spontaneous revertant to sensitivity from the C1 clone. Previously we presented data on the sets of genes whose expression is altered in these cell clones after 20 h exposure to dexamethasone (Dex). The two sensitive clones, which respond by undergoing apoptosis starting about 24h after Dex is added, both showed >2.5-fold induction of 39 genes and 2-fold reduction of expressed levels from 21 genes. C1-15, the resistant clone, showed alterations in a separate set of genes. In this paper, we present further analysis of the data on genes regulated in these cell clones after 20 h Dex and compare them with the genes regulated after 12h Dex. Some, but not all the genes found altered at 20 h are altered at 12h, consistent with our hypothesis that sequential gene regulation eventually provokes full apoptosis. We also compare the levels of basal gene expression in the three clones. At the basal level no single gene stands out, but small sets of genes differ >2-fold in basal expression between the two sensitive and the resistant clone. A number of the genes basally higher in the resistant clone are potentially anti-apoptotic. This is consistent with our hypothesis that the resistant cells have undergone a general shift in gene expression.

Duration of Rhinovirus Shedding in the Upper Respiratory Tract in the First Year of Life

BACKGROUND: Current molecular diagnostic methods have detected rhinovirus RNA in a high proportion of asymptomatic infants and children, raising the question of the clinical significance of these findings. This study investigates the prevalence of prolonged rhinovirus RNA presence in the upper respiratory tract of infants during the first year of life. METHODS: In a longitudinal study, infants were followed from birth up to 12 months. Nasopharyngeal specimens were collected monthly (months 1–6 and month 9) and during an upper respiratory infection. Rhinoviruses were detected by quantitative reverse-transcription polymerase chain reaction. Presence of repeated rhinovirus RNA was evaluated by nucleotide sequence analysis. RESULTS: A total of 2153 specimens from 362 infants were studied; 341 distinct rhinovirus infections in 216 infants were identified. Follow-up specimens were available within 30 days for 179 infections, creating the sample set to assess prolonged rhinovirus presence. Of the 179 infections, 46 involved the detection of the same rhinovirus strain in repeated specimens, including 8 events of prolonged presence of the same strain (detected in specimens collected >30 days apart), representing 4.5% of the evaluable rhinovirus infections. There were 26 events in which a rhinovirus strain was replaced by a different strain within a 30-day interval, representing 14.5% of the 179 infections. CONCLUSIONS: Although rhinovirus infections are common in healthy infants, prolonged presence of rhinovirus RNA in the respiratory tract after an upper respiratory infection was uncommon (<5%). Detection of rhinovirus RNA in an infant most likely represents an infection within a 30-day period.

Converting cell lines representing hematological malignancies from glucocorticoid-resistant to glucocorticoid-sensitive: Signaling pathway interactions

Mitogen-activated protein kinases (MAPKs), protein kinase A (PKA) and mTOR pathways modulate the apoptotic effects of glucocorticoids (GCs) in human lymphoblastic leukemia CEM cells. We now show that manipulation of these pathways converts several cell lines, representing other lymphoid malignancies, from GC-resistant to GC-sensitive. Basal levels of phosphorylated JNK and ERK were elevated in the GC-resistant cells. Treatments that directly or indirectly reduced phosphorylated JNK and ERK resulted in Dex sensitivity in five resistant lymphoid cell lines. Sensitivity to GC-driven apoptosis correlated with GC-dependent increases in phosphorylated and total glucocorticoid receptor, and in increased levels of the pro-apoptotic protein Bim.

Acute Otitis Media and Other Complications of Viral Respiratory Infection.

BACKGROUND: Viral upper and lower respiratory tract infections (URI, LRI) are common in infants. We determined the prevalence of viral URI and its complications, including acute otitis media (AOM) and LRI, and assessed the effect of bacterial-viral interactions, and genetic and environmental risks on AOM development. METHODS: Healthy infants were enrolled from near birth and followed to the first episode of AOM up to 12 months of age. Nasopharyngeal specimens were collected at monthly intervals (months 1–6, 9) and during viral URI episodes for bacterial culture and viral polymerase chain reaction studies. Subjects were followed closely for AOM development. RESULTS: A total of 367 infants were followed for 286 child-years; 887 URI (305 infants) and 180 AOM episodes (143 infants) were documented. Prevalence of URI, LRI, and AOM in the first year was 3.2, 0.25, and 0.67 per child-year, respectively. Cumulative AOM incidence by ages 3, 6, and 12 months was 6%, 23%, and 46%. Infants with and without AOM had 4.7 and 2.3 URI episodes per child-year, respectively (P < .002). Pathogenic bacterial colonization rates by month were significantly higher in infants with AOM (P < .005). Breastfeeding reduced both URI and AOM risks (P < .05). Significant bacterial-viral interactions occurred with Moraxella catarrhalis and a variety of respiratory viruses and altered URI and AOM risks. CONCLUSIONS: Almost half of infants experienced AOM by age 1. Important AOM risk factors included frequent viral URI, pathogenic bacterial colonization, and lack of breastfeeding. Bacterial-viral interactions may play a significant role in AOM pathogenesis and deserve further investigation.

Gene expression profiling of leukemic cells and primary thymocytes predicts a signature for apoptotic sensitivity to glucocorticoids

BackgroundGlucocorticoids (GCs) play an integral role in treatment strategies designed to combat various forms of hematological malignancies. GCs also are powerful inhibitors of the immune system, through regulation of appropriate cytokines and by causing apoptosis of immature thymocytes. By activating the glucocorticoid receptor (GR), GCs evoke apoptosis through transcriptional regulation of a complex, interactive gene network over a period of time preceding activation of the apoptotic enzymes. In this study we used microarray technology to determine whether several disparate types of hematologic cells, all sensitive to GC-evoked apoptosis, would identify a common set of regulated genes. We compared gene expression signatures after treatment with two potent synthetic GCs, dexamethasone (Dex) and cortivazol (CVZ) using a panel of hematologic cells. Pediatric CD4+/CD8+ T-cell leukemia was represented by 3 CEM clones: two sensitive, CEM-C7–14 and CEM-C1–6, and one resistant, CEM-C1–15, to Dex. CEM-C1–15 was also tested when rendered GC-sensitive by several treatments. GC-sensitive pediatric B-cell leukemia was represented by the SUP-B15 line and adult B-cell leukemia by RS4;11 cells. Kasumi-1 cells gave an example of the rare Dex-sensitive acute myeloblastic leukemia (AML). To test the generality of the correlations in malignant cell gene sets, we compared with GC effects on mouse non-transformed thymocytes.ResultsWe identified a set of genes regulated by GCs in all GC-sensitive malignant cells. A portion of these were also regulated in the thymocytes. Because we knew that the highly Dex-resistant CEM-C1–15 cells could be killed by CVZ, we tested these cells with the latter steroid and again found that many of the same genes were now regulated as in the inherently GC-sensitive cells. The same result was obtained when we converted the Dex-resistant clone to Dex-sensitive by treatment with forskolin (FSK), to activate the adenyl cyclase/protein kinase A pathway (PKA).ConclusionOur results have identified small sets of genes that correlate with GC-sensitivity in cells from several hematologic malignancies. Some of these are also regulated in normal mouse thymocytes.