Abstract

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Background: Respiratory adenovirus (ADV) infections activate acute phase proteins, so they are often interpreted as bacterial infections. The aim of this study was to monitor indicators of inflammation (C-reactive protein [CRP], white blood cell and neutrophil counts, Interleukin 6 [IL-6] levels, and lipopolysaccharide binding protein [LPB] capacity) for comparison of ADV infections with influenza and other viral respiratory illness in a pediatric population.
Methods: A total of 171 patients between the ages of one month and 16 years were selected for study, of which 106 were ADV-positive, 32 were ADV-negative, and 33 were positive for influenza virus. ADV infection was confirmed via quantitative polymerase chain reaction (PCR) testing of nasopharyngeal swabs for human ADV-DNA.
Results: When compared with ADV-negative and influenza-positive patients, those with ADV infections showed significantly elevated serum CRP concentrations and left-shifted leukocyte counts. IL-6 levels also differed significantly between ADV-positive and ADV-negative patients. Antibiotic therapy did not influence the course of ADV infections.
Conclusions: ADV and bacterial infections may be indistinguishable clinically, necessitating additional and specific diagnostic investigation. In this respect, the judicious use of acute phase proteins (CRP, interleukins) as a differentiating feature of ADV could have merit.

Introduction

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Acute respiratory infections are frequently attributable to respiratory syncytial (RSV) or influenza viruses or rhino-, entero- and adenoviruses. Unlikle-most viral illnesses, however, infection with adenovirus (ADV) is often characterized by high fever of long duration, leukocytosis with neutrophilia and elevated peripheral blood inflammatory indices. Clinically, these parameters may mimic those of bacterial disease, where the inflammatory response is mediated by various cytokines (eg. Interleukin 6 [IL-6]).
In order to determine the degree and frequency of inflammatory marker elevation with ADV Infection, we monitored blood levels of C-reactive protein (CRP) and IL-6 in children with viral symptoms.Children with provend influenza and other nonspecific viral respiratory infections served as controls. Particular attention was paid to white blood cell (WBC) counts and percentages of neutrophils. In select cases radiographs of the lung, throat swabs (for group A streptococci), blood cultures and serum leukotriene levels (IL-6, IL-10, and LBP) were obtained. Nature and duration of fever, antibiotics used and length of hospitalization were also recorded.
Ultimately, the goal of our study was to evaluate the diagnostic relevance of CRP and IL-6 for ADV infection in a pediatric setting compared with influenza and non-ADV viral illness.

Methods

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Patients
All patient data was analyzed retrospectively at the Department of Pediatrics and Adolescent Medicine of Muldentalkliniken, Wurzen. The study population ranged from one month to 16 years of age.Children with ADV infections had been routinely examined and treated in the interval of November 2003 to June 2007, while those with Influenza virus were seen between February and May 2005. Qualifying patients had the following diagnoses:
1). Upper airway infectious diseases (rhinitis, pharyngitis, tonsillitis, laryngitis, conjunctivitis, and otitis), 2). bronchitis or pneumonia, and 3). enteritis. Fever was defined as axillary temperature (C) ≥37,5°or ≥38,5° rectally.
Methods
Human ADV-DNA was quantitatively tested in nasopharyngeal swabs by real-time polymerase chain reaction (PCR) [1]. Serum CRP, WBC counts (Gpt/L), neutrophil counts (Gpt/L) and percentage of neutrophils were determined upon hospital admission. Double infected patients (ADV plus RSV or influenza) were excluded from study. Subjects were also eliminated if positive for group A streptococcal pharyngitis by Strep A-test, bacteremia by blood cultures, urinary tract infections by urin analysis (and bacterial counts with microbial identification as needed) and enteritis by stool cultures. In 26 patients, leukotriene levels (IL-6, IL-10, and LBP) were determined (Fa. Siemens Medical Solutions GmbH, Bad Nauheim, Germany). Chest radiographs were performed on select ADV-positive patients.
Statistical Analysis
Means and quartiles of outcomes were computed. Distributions of frequencies were compared by the nonparametric Related Samples Wilcoxon Signed Ranks Test and by the Independent Samples Mann-Whitney U-Test (nonparametric U-Test). Proportions were evaluated by Fisher’s Exact Test. Statistical significance was set at a p-value

Results

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Of 155 patients with a presumtive diagnosis of ADV infection 123 proved ADV-positive and 32 ADV-negative. Patients with double infections (12 with RSV and five with influenza) were excluded from study. The ratio of male to female patients examined by PCR was 83:53 (1,6:1). For ADV-positive and ADV-negative patients, this ratio was 62:44 (1,4:1) and 23:9 (2,5:1), respectively. Overall, differences by gender were not statisticaly significant. Of the ADV-positive patients, 70% were infants, and 85% of ADV infections occurred during the months of October through March.
Clinical Findings
Within the study population, 72.6% (77/106) of ADV-positive patients showed upper airway symptoms, while 21.7% (23/106) also suffered from otitis media, 51.1% (54/106) from tonsillopharyngitis, and 27.3% (29/106) from conjunctivitis. Radiographs further indicated lower air-way involvement (pneumonia, obstructive bronchitis) in 17.9% (19/106) of patients and 9.4% (10/106) of cases upper airway infections was accompanied by enteritis. For five patients symptomatology was complicated by convulsions.
Of the ADV-negative patients, 68,8% (22/32) had upper airway infection (five with otitis media, four with tonsillopharyngitis, and eight with conjunctivitis). Pneumonia was radiographically confirmed in six cases (18,0%), enteritis in three cases (9,4%), and one patient experienced convulsions. A conjunctival swab was positive for ADV in 2 children. Influenza was diagnosed in 33 patients, all with similar clinical pictures, but none with pneumonia.
Results of ADV-positive (A), -negative (B) and influenza-infected (C) patients for age, nature and duration of fever, length of inpatient stay, CRP levels, WBC counts, LBP, and p-values are shown in Table 1. Of note are statistically significant differences of CRP, WBC count (Gpt/L), neutrophil count (Gpt/L) and age in ADV-positive subjects, compared with influenza-infected patients differences between ADV-positive and ADV-negative patients were also statistically significant with respect to CRP, WBC, neutrophils, IL-6, and LBP.
CRP
In the ADV-positive group, mean CRP elevation was as high as 40.7mg/L (interquartile range: 15.9-70.7mg/L). Given clinical and laboratory data at a CRP threshold of 40mg/L, significantly higher/lengthier fever, older age, and longer inpatient stay, as well as higher WBC and neutrophil counts were associated with ADV-positive patients (Table 2). The percentage of ADV-positive patients with CRP50mg/L. For CRP >100mg/L but (
Interleukins
In ADV-positive patients, IL-6 elevation was significantly higher (p=0.039) compared with ADV-negative status. While respective levels of IL-10 did not differ significantly (Table 1).
Antibiotic therapy
For the CRP threshold 40mg/L, statistical significance was also observed relative to antibiotic therapy (p=0.001) (Table 2). Data at breakpoints of 20mg/L, 40mg/L, 50mg/L, and 80mg/L are shown in Table 4. Of ADV-positive patients, 37.5% (40/106) received antibiotics, but treatment ceased for 60.0% (24/40) when presented with ADV disease confirmation. The remaining 62.0% (66/106) received no antibiotics (p=0.001). CRP and WBC outcomes for antibiotically treated ADV-negative and influenza-infected patients did not differ significantly.
Other laboratory data
Blood cultures were obtained from 14.5% of patients and throat swabs or Strep-A-tests from 25.4%, all with negative results.

Discussion

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Epidemiology, clinical picture, and differential diagnosis
Worldwide figures for ADV infection in children under the age five years range from 2.0%-5.0% for respiratory illness and 5.0%-15.0% for enteric disease. For the most part, these infections are limited to acute, highly febrile tonsillopharyngitis or enteritis, although systemic involment (liver, kidneys, skin, and central nervous system can occur-) and may at times be lethal. The clinical picture of the ADV infection is fairly nonspecific. Children with ADV tonsillopharyngitis have temperatures above 39.0°C for 5-6 days, often with conjunctivitis and otitis media one-third of cases). ADV tonsillitis can be distinguished from a streptococcal source only through decisive documentation [2-8]. Data from our laboratory-relative to patient age, nature and duration of fever, and length of therapy for ADV-positive, ADV-negative, influenza-infected, and RSV-positive patients did not differ substantially from the medical literature consensus.
During the last decade of the twentieth century it was generally accepted that CRP elevation served to differentiate viral and bacterial infections. Viral infections were, in essence, excluded by high levels of CRP, especially with lower respiratory tract disease [9]. A prior study [10] showed that in 45% of ADV infections, bacterial infection was concurrent.This was, in fact, the case overall in 67% of illnesses, such as pneumonia or acute otitis media. The authors thus argued that patients with mixed viral and bacterial infections should be treated with antibiotics. Nevertheless, on the basis of other recent research and our results, this tenet cannot be undisputed. Neither anamnestic and clinical data nor the combination of Erythrocyte sedimentaion rate, CRP and leukocyte count can be used unreservedly for a differentiation between bacterial and viral (in particular ADV) infection [11,12].
CRP and ADV
Todate, Korppi and Kroger [9] found that in12% of 99 patients with serologically confirmed viral infection, CRP levels exceeded 40mg/L, and 5% had CRP levels >80mg/L. This finding clouds the ability of CRP testing to differentiate viral from bacterial infections. Studies addressing the role of CRP in ADV infections are few-, but do indicate that CRP levels are elevated in this setting. When Appenzeller et al [3] tested 87 children with ADV infection and 130 children with influenza, they accrued median CRP levels of 49mg/L and 9mg/L, respectively (p=0.001). Similarly, investigations led by Chuang [5] and Dominguez [13] found CRP levels of 68.3mg/L (8.0-196.7mg/L) and 44.0±35mg/L (25.5%>70mg/L), respectively with ADV infection. Farng et al [6] also reported a CRP of 67.1± 49.9mg/L, for ADV-infected children, which was significantly higher (pp
Above efforts by Appenzeller [3] and Chuang [5] excluded bacterial superinfections based on culture-negative blood samples, urine specimens, and throat swabs, which our results support. Ultimately, CRP values exceeded 50mg/L 42.8% of our ADV-positive patients; and nine of 11 patients with CRP values >100mg/L but200mg/L. This corresponds well with related publications. In the Appenzeller study [3], 76% of ADV-infected patients had CRP values >100mg/L, while other studies of ADV-positive patients have cited 19.0% with CRP >50.0mg/L [14] and 38.5% at levels >60.0mg/L[4]. Nakayama et al [16] posted a mean CRP value of 19.0mg/L with ADV infection, versus40mg/L [7]. The sol agent implicated in observed WBC and CRP elevations has been ADV [15].
Leukocytes and ADV
The high WBC and neutrophils counts we obtained in patients positive for ADV by PCR have been corroborated by other authors [3,4,7,13-15]. In ADV infection, compared with influenza and RSV-disease or with ADV-negative status, these parameters are significantly elevated [3,5,13].
Antibiotics and ADV
The fact that CRP elevation and a high WBC count are usually hallmarks of bacterial infections explains why antibiotics are often initiated with ADV infections. By CRP breakpoint (≤50.0mg/L or >50.0mg/L), 25.0% versus 60.0% of patients, respectively were treated with antibiotics in the Appenzeller study (p=0.001)[3], while 41.0% to 71.0% of patients were given antibiotics in other studies, without evidence of bacterial superinfection [5,7,8,18]. Kawasaki [14] effectively excluded bacterial superinfection in their patients with ADV infection. Blood cultures had been obtained in 38 patients (44%), cerebrospinal fluid cultures in 15 (17%), urine cultures in 65 (755) and throat antigen tests for detection of group A streptococci in 16 (18%) [3].
Of our ADV-positive patients, 37.7% (40/106) received antibiotic therapy, although in 60.0% (24/40), therapy ceased without consequence upon PCR confirmation. This experience is aligned with another study of ADV serotype 3-infections [7], in which 64.0% of 107 hospitalized children went without antibiotics, and outcomes were excellent. Reports of ADV infection complicated by bacterial superinfection, as occurs with influenza, parainfluenza, and RSV [19, 20] are scarce. A-One-study from 1976 [21] reported combined viral and bacterial infections in 40% of patients, with a quarter of the cases involving ADV. Another report by Ellenbogen et al [22] detailed ADV infection accompanied by bacterial pneumonia but devoid of causal relationship. Additionally, there is an account [17] that 2 of 46 ADV-positive children acquired superinfections, one a urinary tract infection with E. coli, and the other a bacteremia due to Moraxella catarrhalis. Severien et al [23] also described a patient with simultaneous ADV and B. pertussis infection, and several case studies of staphylococcal pneumonia after serologically confirmed ADV infections are in print [24, 25]. Despite best efforts, however, no recent publications surfaced dealing with local bacterial superinfection (confirmed by blood culture or a related method) after PCR- or culture-verified ADV infection.
Cytokines and ADV
Current knowledge of ADV-associated inflammation is seemingly incomplete. In 1994 -, however, Mistchenko et al - [26] published a study of ADV-infected children, from whom various cytokine determinations (IL-6, IL-8, and TNF-alpha) were obtained. They concluded that the magnitude of elevations in these parameters correlated with desease severity. Higher TNF-gamma levels have been found in ADV-positive children, compared with RSV-infected children and with control subjects. IL-6 levels also appear significantly higher in patients with ADV infections (p
In our ADV-positive group, IL-6 levels were significantly higher compared with ADV-negative subjects, thus implicating acute-phase reactants in ADV infections, and our results conform with those of other investigators [28 29]. During the progression of a lethal type 3-ADV pneumonia, elevations of lactate dehydrogenase, ferritin, interferon-gamma, and IL-6 were recorded [30]. Significantly higher IL-6 concentrations have also been demonstated in the nasal mucus of ADV-infected patients compared with asymptomatic controls [31]. An experimental study with gene therapy similarly showed that mononuclear cells, once -, incubated with ADV,
have the capacity to activate proinflammatory cytokines [32].
Until recently, the phenomenon of elevated acute phase proteins (CRP, IL-6) in ADV infection, as well as the significantly higher WBC and neutrophil counts, was not fully understood. Due likely to an ADV trigger, the leukotriene cascade and induction of a Th1- type immune response take place, followed by a rapid increase of acute phase proteins [33]. Increased concentrations of IL-8 and TNF-alpha have also variably been found with ADV infections. Vacillations in IL-6 have correlated significantly with clinical signs of ADV, disease severity, and therapeutic outcome [26]. It is thus possible to explain baseline as well as abnormal elevations of inflammatory mediators in ADV infections.
Because blood levels of procalcitonin (PCT) can help diffentiate viral and bacterial infections and gauge the severity of bacteriemia, PCT levels, in our view, are recommended for assessing ADV respiratory infections of children. At present, there is no discernible evidence base for this test. However, studies addressing the diagnostic difficulties of ADV infections are in short supply [34].

Conclusion(s)

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Further research of paediatric ADV infections is warrented, particularly with respect to the role of cytokines and relevant diagnostic aids. Treatment of presumptive bacterial (super-) infection, based entirely on elevations of CRP and WBC counts, is no longer justified. There is ample evidence to show that ADV infection alone may incite acute phase proteins, rendering antibiotic treatment unnecessery.

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Competing Interests

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