Comparison of the FilmArray Respiratory Panel and Prodesse real-time PCR assays for detection of respiratory pathogens.

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.

A single intramuscular dose of ceftriaxone changes nasopharyngeal bacterial flora in children with acute otitis media.

The increasing prevalence of drug-resistant bacteria is attributed to the extensive use of antibiotics, which causes selective pressure on the nasopharyngeal bacterial flora. Shortened courses of antibiotics have been proposed to decrease the development of resistant strains. We determined the effect of a single intramuscular dose of ceftriaxone (50 mg/kg) on the nasopharyngeal bacterial flora in 167 children (median age 13 mo) with acute otitis media. Nasopharyngeal samples for bacterial culture were obtained before and 5 d after treatment with ceftriaxone. Before treatment, Moraxella catarrhalis was isolated in 99 (59%) children, Streptococcus pneumoniae in 87 (52%), and Haemophilus influenzae in 53 (32%). After treatment, M. catarrhalis was found in 62 (37%) children, which constitutes a 37% decrease in the colonization rate by this pathogen (p < 0.001). S. pneumoniae was isolated in 50 (30%; 43% decrease) and H. influenzae in 17 (10%; 68% decrease) children after treatment (p < 0.001 for both). Before treatment, 60% of pneumococcal isolates were sensitive to penicillin, 26% were of intermediate susceptibility, and 14% were penicillin-resistant. Eradication of S. pneumoniae occurred mainly in children with penicillin-sensitive isolates. As a consequence, only 24% of pneumococcal isolates that remained after treatment were sensitive to penicillin, 59% were penicillin-intermediate, and 16% were penicillin-resistant. A single dose of ceftriaxone resulted in significant changes in the nasopharyngeal bacterial flora, increasing the relative prevalence of pneumococcal strains with decreased susceptibility to penicillin.

Viral-Bacterial Synergy in Otitis Media: Implications for Management.

Despite the extensive use of broad-spectrum antibiotics, poor clinical response to the treatment of acute otitis media is common. Evidence derived from numerous studies during the past two decades supports a crucial role for respiratory viruses in the etiology and pathogenesis of acute otitis media, and recent studies indicate that viruses may also have a profound adverse effect on the resolution of this disease. Viruses seem to interact with bacteria and enhance the local inflammatory process in the middle ear. Effective adjuvant therapies are needed to improve outcome in children with otitis media. Increasing knowledge of the role of viruses, viral-bacterial interaction, and host inflammatory mechanisms in otitis media may lead to major improvements in the management of this disease.

Increasing importance of viruses in acute otitis media.

Acute otitis media is generally considered a simple bacterial infection that can be effectively treated with antibiotics. However, despite the extensive use of broad-spectrum antibiotics, poor clinical response to treatment of acute otitis media is common in children. Numerous studies ranging from animal experiments to extensive clinical studies have clearly demonstrated that respiratory viruses play a crucial role in the aetiology and pathogenesis of acute otitis media. Viral infection of the upper respiratory tract initiates the whole cascade of events that finally leads to the development of acute otitis media as a complication. Respiratory viruses induce a release of inflammatory mediators in the nasopharynx, increase bacterial colonization and adherence, and have a suppressive effect on the host's immune defense. Recent data indicate that at least some types of viruses actively invade the middle ear. Viruses also seem to enhance the inflammatory process in the middle ear and impair the outcome of the disease. Vaccines against the major viruses predisposing to acute otitis media hold a great promise for the prevention of this disease. Major advances in the management of acute otitis media will require further research into the mechanisms of viral infection, viral-bacterial interaction and the host inflammatory response during viral infection.

Free secretory component as a standardization protein for nasopharyngeal specimens from children with upper respiratory tract infection.

Free secretory component (FSC) has been recommended as a reliable protein for correction of the unknown dilution in tracheal aspirate samples from preterm infants. To investigate whether FSC would also provide a valid standardization protein for samples of nasopharyngeal secretions, this study determined the intersubject variation and the alteration over time in the concentrations of FSC in nasal secretions from 35 children (median age 14 months) who participated in an antibiotic efficacy trial. Nasopharyngeal aspirates were obtained at enrolment and after 2-3 d. FSC in the specimens was quantified by a direct enzyme immunoassay. The concentrations of FSC in the nasal secretions ranged from 0.08 to 189.6 microg ml(-1) (median 12.3 microg ml(-1); the ratio of the highest to the lowest concentrations was 2370, the difference between the 90th and 10th percentile concentrations was 189-fold and the difference between the 75th and 25th percentile values was 26. FSC concentrations were significantly lower in children aged < or =12 months (median 2.2 microg ml(-1) than in the older children (median 21.5 microg ml(-1); p = 0.035). Between the first and the follow-up specimens, 65% of the children had > or =2-fold difference in the levels of FSC in the secretions. Because an optimal standardization protein should show minimal variation between individuals and over time, FSC may not be a suitable protein for correction of the unknown dilution of nasopharyngeal specimens from children with upper respiratory tract infection.

Quantification of cytokines and inflammatory mediators in samples of nasopharyngeal secretions with unknown dilution.

In the study of inflammatory mechanisms in the upper respiratory tract, the unknown dilution of collected samples of nasal secretions poses a serious problem for interpretation of the measured concentrations of various substances in the specimens. We investigated the magnitude of the dilution problem in a true clinical research situation and determined the validity of using the levels of total protein, albumin, and secretory IgA in nasal secretions to correct for the unknown dilution. The study samples consisted of simultaneously obtained nasopharyngeal aspirates and nasal lavage specimens from 52 children with upper respiratory tract infection. The dilution factors of the nasal lavage specimens varied widely between 1.8 and 432 (median, 11.2). Of the three proteins studied, total protein had the narrowest inter-subject variation in the nasal secretions of the children and thus seemed to provide the best standardization method for comparing levels of substances between individuals. Concentrations of IL-6 standardized with total protein correlated significantly better with the true IL-6 concentrations in the nasal secretions than did IL-6 levels measured in the nasal lavage specimens without standardization (p = 0.049). These findings suggest that the most common current practice of measuring substances in nasopharyngeal specimens, i.e. measuring without correction for the dilution, may produce "false-negative" results. Potentially important information on inflammatory mechanisms may be undetected if false-negative results mask real differences between groups. The use of exogenous markers of dilution might improve the accuracy of quantifying substances in nasal secretions.

Prevalence of various respiratory viruses in the middle ear during acute otitis media.

BACKGROUND: Vaccines against respiratory viruses may be able to reduce the frequency of acute otitis media. Although the role of respiratory viruses in the pathogenesis of acute otitis media is well established, the relative importance of various viruses is unknown. METHODS: We determined the prevalence of various respiratory viruses in the middle-ear fluid in 456 children (age, two months to seven years) with acute otitis media. At enrollment and after two to five days of antibiotic therapy, specimens of middle-ear fluid and nasal-wash specimens were obtained for viral and bacterial cultures and the detection of viral antigens. The viral cause of the infections was also assessed by serologic studies of serum samples obtained during the acute illness and convalescence. RESULTS: A specific viral cause of the respiratory tract infections was identified in 186 of the 456 children (41 percent). Respiratory syncytial virus was the most common virus identified in middle-ear fluid: it was detected in the middle-ear fluid of 48 of the 65 children (74 percent) infected by this virus (P< or =0.04 for the comparison with any other virus). Parainfluenza viruses (15 of 29 children [52 percent]) and influenzaviruses (10 of 24 children [42 percent]) were detected in the middle-ear fluid significantly more often than enteroviruses (3 of 27 children [11 percent]) or adenoviruses (1 of 23 children [4 percent]) (P< or =0.01 for all comparisons). CONCLUSION: Respiratory syncytial virus is the principal virus invading the middle ear during acute otitis media. An effective vaccine against upper respiratory tract infections caused by respiratory syncytial virus may reduce the incidence of acute otitis media in children.

Serum interleukin-6 in bacterial and nonbacterial acute otitis media.

BACKGROUND: Increasing prevalence of antibiotic-resistant bacteria is a serious clinical problem that calls for reduction of unnecessary use of antibiotics. Acute otitis media (AOM) is the most common reason for antibiotic therapy in the United States. Approximately 30% of AOM cases do not have a bacterial etiology. Rapid identification of these cases could help withhold unnecessary antibiotic treatment. OBJECTIVE: To determine the usefulness of serum levels of interleukin-6 (IL-6), an acute phase cytokine shown to be a reliable marker of neonatal bacterial infection, in differentiation between bacterial and nonbacterial AOM in children. STUDY DESIGN: IL-6 was measured in stored serum samples from 184 children (mean age, 22 months) with AOM who were enrolled in antibiotic efficacy trials at our department. The samples were obtained at enrollment and at 9 to 12 days after initiation of antibiotic therapy. Sera from 21 uninfected children (mean age, 23 months) were used as controls. The etiology of AOM was determined by bacterial and viral cultures as well as respiratory syncytial virus antigen detection in the middle ear fluids obtained by tympanocentesis. RESULTS: Bacterial etiology of AOM was confirmed in 125 children (68%), whereas in 59 children (32%) no bacterial pathogen could be detected in the middle ear fluid. Children with bacterial AOM had significantly higher IL-6 levels than those with nonbacterial AOM (median, 11.5 vs 3.7 pg/mL). However, this difference was almost entirely attributable to pneumococcal AOM specifically. IL-6 levels in children with AOM caused by Streptococcus pneumoniae were significantly higher (median, 40.1 pg/mL) than in AOM caused by Haemophilus influenzae (7.3 pg/mL) or Moraxella catarrhalis (6.8 pg/mL). At the cutoff value of 30 pg/mL, the specificity of IL-6 for detection of pneumococcal AOM was 91% with a sensitivity of 61%, but its sensitivity for detection of bacterial AOM in general was only 27%. CONCLUSIONS: Low levels of IL-6 do not rule out bacterial etiology of AOM in general; therefore, IL-6 is not sensitive enough as a marker of bacterial AOM. Surprisingly, serum IL-6 levels in pneumococcal AOM were significantly higher than the levels associated with other bacterial AOM, and serum IL-6 levels of >30 pg/mL were highly specific for pneumococcal AOM. These findings suggest a distinctive role for S pneumoniae in the pathogenesis of AOM.

Use of monoclonal antibodies to identify serotypes of enterovirus isolates.

Nonpoliovirus enteroviruses cause a variety of diseases that are common in young children and adults. The "gold standard" for laboratory diagnosis of enteroviruses is cell culture isolation, followed by serotype identification by neutralization assay. These procedures are time-consuming and expensive. Rapid serotype identification of enteroviruses is important in differentiating nonpoliovirus enterovirus pathogens from vaccine strain polioviruses that can be shed for some time after vaccination. In the present investigation, we evaluated a rapid method for serotype identification of enteroviruses by indirect immunofluorescence assay (IFA) using commercially available monoclonal antibodies for polioviruses, coxsackieviruses type B, and six serotypes of commonly circulating echoviruses. Of 291 isolates of enteroviruses included in the study, 95 were polioviruses and 196 were nonpoliovirus enteroviruses. Two hundred thirty-four of these (38 polioviruses and 196 nonpoliovirus enteroviruses) were consecutively grown in the laboratory over a 5-year period. IFA identified the serotypes of 74% of the consecutive isolates and 71% of all enterovirus isolates by yielding a positive staining result. The levels of agreement in the identification of the enterovirus group between IFA and neutralization tests were 92% for consecutively grown isolates and 85% for all enterovirus isolates. The sensitivity of the IFA for the detection of viruses for which specific monoclonal antibodies were applied was 73% for polioviruses, 85% for coxsackieviruses type B, and 94% for echoviruses. Specificity was near 100% for polioviruses and coxsackieviruses type B and 94% for echoviruses. We conclude that IFA can be helpful as a preliminary test for serotype identification of enteroviruses. The results are most accurate when the test identifies the isolate as a poliovirus.

Amoxicillin middle ear fluid penetration and pharmacokinetics in children with acute otitis media.

BACKGROUND: Acute otitis media (AOM) is a common childhood infectious disease. The efficacy of antibiotic dosing regimens is usually assessed by antibiotic plasma pharmacokinetics or middle ear fluid (MEF) concentration at one or two time points. Viral coinfection in AOM reduced antibacterial efficacy of antibiotics. OBJECTIVE: To determine amoxicillin MEF penetration and pharmacokinetics in bacterial and combined bacterial and viral AOM. METHODS: Thirty-four children with AOM were enrolled, and MEF was collected by tympanocentesis for bacterial culture and viral studies. Nasal wash and venous blood were also obtained for viral culture and serologic studies, respectively. Subjects were treated with amoxicillin 40 mg/kg/day orally, divided in equal doses every 8 h. During the second visit (48 to 72 h later) the subjects, with the regular morning amoxicillin dose withheld, were given an oral amoxicillin dose of 25 mg/kg. Thereafter two blood samples and one MEF sample by tympanocentesis were collected from each child at selected times between 0.5 and 4.0 h after dosing for bacterial and viral studies and amoxicillin concentration determination by high performance liquid chromatography. RESULTS: Eleven (37%) children had only bacterial infection, 6 (20%) had viral infection only, 6 (20%) had both bacterial and viral infections and in 7 (23%) neither bacterial nor viral pathogens were recovered. MEF bacterial culture was positive in 23 of 40 ears (57.5%) before treatment with amoxicillin (40 mg/kg/day) and was still positive in 4 of 38 ears (10.5%) after 2 to 3 days of treatment. Amoxicillin plasma concentration reached its peak at 1.0 to 1.5 h after a 25-mg/kg oral dose. The estimated MEF concentration peak occurred 3.0 h after the dose with MEF concentrations ranging from undetectable to 20.6 microg/ml and a mean of approximately 9.5 microg/ml. Geometric mean amoxicillin concentrations were lowest in virus-infected children (2.7 microg/ml), nearly the same in culture-negative samples from children without viral infection (2.9 microg/ml), higher in children with combined bacterial and viral infection (4.1 microg/ml) and highest in children with bacterial-only infection (5.7 microg/ml). CONCLUSIONS: MEF amoxicillin penetration tended to be lower in children with viral infection. The current amoxicillin dosing recommendation of 40 mg/kg/day in three divided dose is inadequate to effectively eradicate resistant Streptococcus pneumoniae, particularly during viral coinfection. A dosing regimen of 75 to 90 mg/kg/day is recommended for AOM.

Role of viruses in middle-ear disease.

Taken together, there is ample evidence suggesting a role for viruses in the pathogenesis of OM. This evidence comes from numerous studies performed in animals and in vitro cell cultures, as well as in adults and children with URI and OM. Viruses induce host immune and inflammatory responses that result in pathology of the ET and the middle ear, and predispose the host in various ways to secondary bacterial infection. A suggested mechanism for the pathogenesis of OM following respiratory viral infection is presented in Figure 1.

Role of leukotriene B4 and interleukin-8 in acute bacterial and viral otitis media.

In order to evaluate the role of polymorphonuclear leukocytes (PMNs) in acute otitis media (AOM), levels of leukotriene B4 (LTB4), a potent inflammatory product of PMNs, and interleukin-8 (IL-8), a PMN chemotactic cytokine, were measured in 271 middle ear fluid (MEF) samples from 106 children with AOM. Forty-two percent of the patients had evidence of respiratory viral infection. At the time of diagnosis, levels of both LTB4 and IL-8 were higher in the MEFs from patients with AOM associated with bacterial or bacterial and viral infection than those MEFs containing no pathogen (p < .05). Antibiotic treatment was not associated with a significant change in levels of LTB4 or IL-8 in the MEFs obtained 2 to 5 days into treatment, compared to those obtained at diagnosis. Bacteriologic failure after 2 to 5 days of treatment was associated with high LTB4 levels in the initial MEFs (p = .05). Recurrence of AOM within 1 month was associated with high IL-8 levels in the initial MEF (p = .04). Our findings suggest that LTB4 and IL-8 are produced during acute infection of the middle ear, and these PMN-related inflammatory substances may play an important role in delaying recovery or in recurrence of AOM. Effective treatment of AOM may require eradication of bacteria by antibiotics, as well as pharmacologic agents that modulate PMN functions.

Interleukin-1 alpha mediates the enhanced expression of intercellular adhesion molecule-1 in pulmonary epithelial cells infected with respiratory syncytial virus.

The mechanisms of virus-induced enhancement of intercellular adhesion molecule-1 (ICAM-1) expression in epithelial cells are unknown. In the present study, the effect of respiratory syncytial virus (RSV) infection on the expression of ICAM-1 in human pulmonary type II-like epithelial (A549) cells was evaluated. Conditioned RSV media (cRSV) produced from growth of RSV in A549 cells induced a significant increase in the expression of ICAM-1. Treatment of the cells with noninfectious cRSV prepared by ultraviolet (UV) irradiation (UV-cRSV) or ribavirin treatment resulted in the expression of ICAM-1 to a similar extent as infectious cRSV. These results suggested that RSV induces the synthesis of a soluble mediator(s) that regulates the expression of ICAM-1. Cytokine analysis by immunoassay and polymerase chain reaction showed that RSV induces the synthesis of interleukin (IL)-1 alpha and -beta, and tumor necrosis factor alpha (TNF-alpha). Preincubation of UV-cRSV with soluble IL-1 receptor (sIL-1r) almost completely blocked the enhancement of ICAM-1 expression. Furthermore, simultaneous incubation of infectious purified RSV with sIL-1r resulted in a significant reduction in enhancement of ICAM-1 expression. Preincubation with neutralizing antibodies to IL-1 alpha and -beta, and TNF-alpha showed that the predominant ICAM-1 enhancing soluble mediator in UV-cRSV was IL-1 alpha. These experiments provide direct evidence for an autocrine mechanism of enhanced ICAM-1 expression in RSV-infected epithelial cells that is mediated primarily by IL-1 alpha. Pulmonary epithelial cells may play an important immunoregulatory role in the microenvironment of the lower respiratory tract infected with RSV.

Bacteriologic failure of amoxicillin-clavulanate in treatment of acute otitis media caused by nontypeable Haemophilus influenzae.

OBJECTIVE: To evaluate the rate of bacteriologic failure of amoxicillin-clavulanate in the treatment of acute otitis media (AOM) and to identify the risk factors associated with failure. METHODS: Ninety-nine subjects (mean age, 21.4 months) with AOM were treated with amoxicillin-clavulanate in two prospective study trials that compared efficacy of two experimental antibiotics with amoxicillin-clavulanate. Tympanocentesis for microbiologic studies was performed in all subjects at enrollment; at 3 to 6 days, during amoxicillin-clavulanate therapy; and at other times when clinically indicated. The subjects were followed up for 1 month. Clinical, bacteriologic, and virologic characteristics of the subjects were analyzed. RESULTS: Bacteriologic failure of treatment occurred in none of 39 subjects (0%) with Streptococcus pneumoniae, two of 25 (8%) with Moraxella catarrhalis, and 11 of 29 (38%) with nontypeable Haemophilus influenzae (NTHi) infection. The failure rate for NTHi was higher than that for other pathogens (p = 0.0007) and was increased when compared with the preceding study period (p = 0.017). Bacteriologic failure was also associated with clinical failure (p = 0.041). In subjects with AOM caused by NTHi the rates of adequate drug compliance were comparable in both success and failure groups. Antimicrobial susceptibility testing by minimum inhibitory concentration and minimum bactericidal concentration (MIC/MBC) assays showed that amoxicillin-clavulanate resistance was not significantly associated with bacteriologic failure of treatment. However, in two subjects, MIC/MBC of the NTHi isolates during therapy were higher than MIC/MBC of the isolates before therapy; these strains of isolates pretherapy and during therapy were discordant as determined by outer membrane protein analysis. The bacteriologic failure rate was higher in nonwhite boys (p = 0.026) and in subjects with a history of three or more previous episodes of AOM (p = 0.008). Other factors such as age, bilaterality of disease, polymicrobial infection, and biotype pattern of NTHi were not associated with treatment failure. When children with adequate drug compliance were analyzed separately, only those with concomitant viral infection of the nasopharynx or middle ear were found to be at an increased risk of bacteriologic failure of treatment (p = 0.04). CONCLUSIONS: The bacteriologic failure rate of amoxicillin-clavulanate therapy for AOM caused by NTHi was higher in the current study period than in the preceding period. Factors contributing to treatment failure were race, gender, proneness to otitis, and concomitant viral infection.

Use of C-reactive protein in differentiation between acute bacterial and viral otitis media.

OBJECTIVES: The objectives of this investigation were: (1) to determine degree of elevation of serum C-reactive protein (CRP) in uncomplicated acute otitis media (AOM); (2) to compare serum CRP levels in bacterial and viral otitis media; and (3) to determine whether a single serum CRP level, obtained early in the course of AOM, could be used to differentiate between viral and bacterial otitis media. DESIGN AND METHODS: Sera were obtained from otherwise healthy infants and children with AOM who were 3 months to 7 years of age between 1989 and 1991. Tympanocentesis, bacterial and viral studies of the middle ear fluids, virologic studies of nasal wash specimens, measurements of serum antibody titers to respiratory viruses, blood counts, and quantitation of serum CRP concentrations were performed. After the initial tympanocentesis, an oral antibiotic was given for the next 10 days. The patients were clinically reevaluated over next 4 weeks. OUTCOME MEASURES: Serum CRP concentrations were compared among subjects with AOM who were divided into four groups based on the results of bacteriologic and virologic studies: group I, Bacterial infection (n = 82); group II, bacterial and viral infections (n = 69); group III, viral infection (n = 12); and group IV, no identifiable pathogen (n = 22). RESULTS: There was no statistical difference in serum CRP values among the four groups. The ranges of CRP were less than 0.6 to 22.8, less than 0.6 to 17.8, less than 0.6 to 2.0, and less than 0.6 to 6.8 mg/dL in groups I through IV, respectively. However, when CRP values in bacteria-positive cases were compared with CRP concentrations in bacteria-negative cases (1.58 +/- 3.16 vs 0.64 +/- 1.24 mg/dL), the difference was statistically significant. Furthermore, a significantly higher proportion of bacteria-positive cases had serum CRP concentrations greater than 2 mg/dL, compared with those in bacteria-negative cases. There was no correlation between initial CRP values and clinical findings and/or the clearance of bacteria from the middle ear. After 10 days of antibiotic treatment, CRP values returned to normal (< 0.6 mg/dL) in all cases. CONCLUSION: In AOM, the range of serum CRP varied from less than 0.6 to 22.8 mg/dL. High CRP values (> 2.0 mg/dL) were associated with 22% of cases of bacterial AOM but only with 6% of nonbacterial AOM. High levels of serum CRP were found to be very specific in detecting bacterial AOM, and no cases of viral AOM without a concurrent bacterial infection were found to exhibit high serum levels of CRP.