PEDIATRICS Vol. 119 No. 6 June 2007, pp. 1069-1075 (doi:10.1542/peds.2006-3294)
ARTICLE |
Temporal Relationships Between Colds, Upper Respiratory Viruses Detected by Polymerase Chain Reaction, and Otitis Media in Young Children Followed Through a Typical Cold Season
a Departments of Otolaryngology
c Pediatrics, University of Virginia Health System, Charlottesville, Virginia
b Department of Otolaryngology, Children's Hospital of Pittsburgh and University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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INTRODUCTION. Otitis media is a frequent complication of a viral upper respiratory tract infection, and the reported co-incidence of those diseases increases with assay sensitivity and sampling density. We determined the incidence of otitis-media complications in young children when referenced to cold-like illnesses and to concurrent virus recovery from the nasopharynx.
METHODS. A total of 60 children from 24 families were followed from October 2003 through April 30, 2004, by daily parental recording of illness signs, weekly pneumatic otoscopic examinations, and periodic polymerase chain reaction assay of collected nasal fluids for common viruses.
RESULTS. One hundred ninety-nine cold-like illnesses were observed, but a sample for virus assay was not collected concurrent with 71 episodes. Of the remainder, 73% of cold-like illnesses were temporally related to recovery of 1 or a combination of the assayed viruses, with rhinovirus predominating. For non–cold-like illness periods, 54 (18%) of 297 assays were positive for virus, and the virus frequency distribution was similar to that for cold-like illnesses. There were 93 diagnosed otitis-media episodes; 65 (70%) of these occurred during a cold-like illness. For the 79 otitis-media episodes with available nasal samples, 61 (77%) were associated with a positive virus result. In this population, the otitis-media complication rate for a cold-like illness was 33%.
CONCLUSIONS. A cold-like illness was not a prerequisite for polymerase chain reaction detection of viruses in the nose and nasopharynx of young children. Viral detection by polymerase chain reaction in the absence of a cold-like illness is associated with complications in some subjects. Otitis media is a complication of viral infection both with and without concurrent cold-like illnesses, thus downwardly biasing coincidence estimates that use cold-based illnesses as the denominator.
Key Words: colds • children • otitis media • virus
Abbreviations: vURI—viral upper respiratory tract infection • OM—otitis media • CLI—cold-like illness • PCR—polymerase chain reaction • RSV—respiratory syncytial virus • cDNA—complementary DNA • TCID—tissue culture infectious dose
It is commonly accepted that viral upper respiratory tract infection (vURI) is causally related to the development of new episodes of otitis media (OM).1–3 However, disagreements exist as to the relative importance of the different viral species in promoting the complication, true complication rate, and relative percentage of all OM episodes that are complications of a vURI.2,4–8 Resolution of these issues is important given that many believe OM to be preventable by using strategies to reduce vURI burden and/or to decouple OM as a presentation.9–11
Typically, the presence of a vURI is recognized based on a set of self-appreciated and/or secondarily assigned symptoms and signs that define a cold-like illness (CLI).12 Because vURIs without symptom/sign expression do not present clinically, co-incidence assessments for the different vURI complications are referenced usually to CLIs. For OM, past estimates suggest that 
20% to 40% of CLIs in children are complicated by OM and that 50% of all new OM episodes are attributable to a CLI.3,8,13 Recent studies show that these estimates are increased by increasing the density of otologic assessments (ie, capturing more OM events).14 Moreover, experimental infection of adults with different upper respiratory viruses shows that one half of those who are judged to be infected by shedding and/or seroconversion do not express signs/symptoms of a CLI.15–19 In 1 analysis of those data, the frequency of otologic complications in non-ill subjects remained significant,18 and other studies showed that preexisting antibodies and antiviral treatment differentially affected the CLI and otologic complications.20,21 Also, the presence of viruses in the nose and nasopharynx without a concurrent CLI was documented in control populations of infants, children, and adults,22–25 although the significance of this observation to presentations of complications such as OM was not assessed.
The purpose of this study was to determine whether subclinical vURIs are complicated by OM in a group of young children followed by daily diary for illness signs, weekly pneumatic otoscopic examinations by study personnel, and periodic collection of nasal secretions for virus assay by polymerase chain reaction (PCR). The null hypotheses tested are that the OM complication rate for a vURI estimated from CLI is an accurate estimate of the true complication rate for vURIs and that the ratios of OM coincidence to total OM episodes are similar when coincidence is defined for CLIs and vURIs. Acceptance or rejection of these hypotheses is important to the design of strategies for preventing OM on the basis of interfering with virus acquisition, virus shedding, or illness presentation during a vURI by providing estimates of the maximum efficacies and efficiencies of different proposed interventions.11
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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The data for this report were abstracted from those available for the first year of our ongoing, 5-year study entitled "Role of Virus and Genetic Susceptibility in Otitis Media." In the study, families with 2 children aged 1 to 5 years are recruited by newspaper advertisement for participation in each year. Exclusion criteria include the presence in either child of a serious medical condition, a medical condition that predisposes to persistent OM, a nonintact or structurally abnormal tympanic membrane, a preexisting sensorineural hearing loss, or an inability to cooperate sufficiently with the examination and test procedures. After affirmation of willingness to participate and acquisition of written informed consent, families are entered into the study in October and followed through April of the respective study year. Families are reimbursed $100 per month for their participation. The study protocol was approved by the institutional review boards at the University of Pittsburgh and the University of Virginia.
The data for this report include general demographic information on the family obtained at entry, and the results for daily diaries maintained by the mother rating the presence/absence of 6 signs (ie, runny nose, nasal congestion, sore throat, cough, fever, and irritability) and recording the presence/absence of a cold or flu on the basis of their usual diagnostic criteria in each of their children; weekly assessments of middle-ear status in the children by pneumatic otoscopy done at either an "in-home visit" (Pittsburgh) or at the study clinics (Charlottesville) by study personnel; and periodic collection of nasal secretions from the children for virus identification by PCR performed at either an "in-home visit" or at the study clinics by study personnel. The purposes of our analyses were to determine the OM complication rate for a CLI and for times of recovery of an upper respiratory virus, and conversely, the frequencies of all OM episodes that are complications of a CLI or of recovery of an upper respiratory virus.
To define a CLI, we used a previously developed algorithm that reduces the between-rater biases in cold assignment.26 Specifically, the different mothers used a wide variety of sign constellations for assigning a cold or flu, and the algorithm standardizes the definition of a CLI across families. The algorithm first assigns an illness day if the maternal report for that day included 
2 of runny nose, nasal congestion, and cough, or if the parental report included runny nose or nasal congestion given that the previous day was assigned by algorithm to an illness day. Then, illness/nonillness days were linked as child-specific strings for sequential observations beginning in October and extending to April 30, with string elements coded as 0 (nonillness day) or 1 (illness day). For the few times where the string sequences were broken by missing values, a default value of 0 was entered unless the bordering observations indicated illness days wherein a value of 1 was entered. All zero-valued elements were deleted from each string to yield variable-length clustered sequences of 1s, which if at least 3 days in length, was considered to be a CLI, with onset defined as the first observation of an illness day and termination as the first nonillness day in a sequence of at least 5 consecutive nonillness days.
Data for bilateral pneumatic otoscopy performed at each examination were classified dichotomously for each ear as OM absent/present on the basis of the otoscopist's ratings of the tympanic membrane with respect to visibility, condition, position, appearance, color, vascularity, light reflex, and mobility.14 Data were coded for the left and right ears as OM absent/present (0 = absent, 1 = present) without consideration to the concurrent expression of otologic signs/symptoms. For each child, observation days for the right and left ears were linked as temporal strings, with interobservation values assigned the value of the initial observation for the interval. An OM episode was defined as a sequential series of 1s bounded by 0s for a given ear.
We proposed to collect periodic nasal secretion samples at monthly intervals and supplemental samples during periods of "parent-identified" cold episodes and/or otoscopically diagnosed OM. This proved not to be possible because some children resisted sample collection in the absence of free secretions (usually non-CLI periods), the failure in some instances of parents to promptly notify study personnel of a cold diagnosis in their child, the unavailability of families for a study visit during a parent-identified cold (eg, holiday, vacation), and the larger than expected number of parent-identified colds, which required reducing the number of samplings during non-CLI periods for budgetary reasons. A total of 513 samples were collected and assayed for viruses.
The technique for collecting secretion from nose and nasopharynx was described previously.25 Briefly, a Yankauer suction device (Conmed, Utica, NY) connected to a Lukens specimen trap (Sherwood Medical, St Louis, MO) was positioned sequentially in each vestibule to aspirate nasal secretions with application of negative pressure (–90 mmHg) from a suction pump. If visible secretion did not reach the tubing, when tolerated by the subject, 1 to 2 mL of nonbacteriostatic saline was instilled into the nostril with aspiration from the contralateral nostril. Visible secretion in the tubing was washed into the trap with saline.
All collected specimens were frozen at –70°C and shipped in batches to our virology laboratory at the University of Virginia for PCR assay to detect adenovirus, coronavirus, influenza virus, parainfluenza virus, rhinovirus (enterovirus), and respiratory syncytial virus (RSV). These viruses were sought by using a protocol adapted from the commercially available Hexaplex procedure (Prodesse, Inc, Waukesha, WI).27 Viral nucleic acid was extracted from 280 µL of aspirate/wash sample using the QIAmp Viral RNA Mini-Kit (Qiagen, Valencia, CA). Complementary DNA (cDNA) was synthesized by using random hexamers and reverse transcription. Viral nucleic acid was purified and reverse transcribed to cDNA when original samples were thawed for the first time. The quantity of cDNA was dictated by the planned number of different PCR amplifications, because testing of refrozen original samples or purified RNA may give false-negative results.25 Purification of adenoviral DNA by this technique (including the superfluous reverse transcription) was equivalent to extraction by a method for DNA (QIAmp DNA Blood Minikit; Qiagen), which was used.28
PCR was performed with master mix and amplification parameters specific for each virus. Unincorporated primers and deoxynucleoside triphosphates were removed from the PCR products with the QIA Quick PCR Purification Kit (Qiagen). Amplified product was detected with an oligonucleotide probe specific for each virus labeled with horseradish peroxidase, using avidin-coated plates and hybridization buffer included in the commercially available Hexaplex assay (Prodesse). After hybridization at 50°C for 1 hour, the plates were washed, substrate added, the reaction stopped after 10 minutes, and optical density of each well measured at 450 nm on a spectrophotometer. The positive cutoff value was 3 SDs above the mean of water controls.
Picornavirus
The primers, composition of PCR master mix, amplification parameters, and probe for hybridization were detailed (adapted protocol from25). Reverse transcriptase-PCR with this method on log dilutions of a titered pool of type 39 rhinovirus demonstrated that 
1 tissue culture infectious dose (TCID)50/0.1 mL of sample was detected.
Coronavirus
The same PCR master mix25 was used for coronaviruses OC43 and 229E, with primers, probe, and amplification parameters for a sequence of the M gene of the 2 viruses as previously detailed.29 The sensitivity of the assay for 229E was 0.01 TCID50/0.1 mL on the basis of titration in MRC-5 cells (human fetal lung fibroblast cell line).
Parainfluenza 1–3, Influenza A and B
The assay for these viruses was conducted by using Hexaplex reagents obtained from the manufacturer (Prodesse). Amplication of cDNA with a proprietary PCR mix containing primers for the viruses (Supermix, Prodesse) was performed by using cycling parameters detailed by Prodesse. Optical density at 450 nm of 0.4 was the positive cutoff, in accord with the manufacturer's directions.
RSV
Primers, probe, and PCR cycling parameters were based on those described previously.30 The sensitivity of the assay determined by using a pool of RSV titered in MRC-5 cells (kindly provided by Dr Ron Turner) was 0.28 TCID50/280 µL.
Adenovirus
PCR for adenovirus is based on the generic PCR developed by Echavarria and colleagues28,31 for detection of all clinically relevant adenovirus strains. The master mix for PCR contained 0.8 µM of each primer (biotinylated Hex 3 and Hex 4 primers) in 1x AmpliTaq Gold buffer with 2.7 mM MgCL2 and AmpliTaq Gold polymerase (Applied Biosystems, Foster City, CA). The amplification parameters were as detailed in a previous publication.32 The oligonucleotide probe Hex 30 labeled with horseradish peroxidase was used in the hybridization step. The sensitivity of PCR for adenovirus was <1 TCID50/0.1 µL on the basis of titration of an unnumbered adenovirus clinical isolate in A549 cells (kindly provided by Mike Ison, MD, MS).
Results for the 513 assays were cast as strings, and the number of reported assays was reduced to 425 independent assays by linking identical virus detections within a 20-day period but without an intermittent observation of "no detectable virus" as a single virus detection (assay), and by assigning multiple assays for the same CLI and/or OM string to the virus detected during that string or to no virus detected as applicable.
For each child, the CLI strings, virus strings, and the left and right OM strings were aligned on the time axis. First, CLI strings were examined for relationships to viruses, classified into 3 categories: CLIs with associated viruses (viruses immediately preceding or embedded within the string), CLIs with no viruses (containing embedded virus negative observations), and CLIs of unknown etiology (strings for which no virus samples were temporally associated), and counted. CLIs with associated viruses were labeled by the viral species, and CLIs that included both positive and negative virus recoveries were labeled by the recovered virus.
OM episodes (either unilateral or bilateral) were classified into 4 groups: OM as a CLI complication (episodes embedded within or immediately after the CLI, with primary etiology assigned to the associated virus, if any), OM as a complication of a viral infection but without evidence of a coexisting CLI (episode with a positive virus sample during the extant OM period), OM independent of a CLI/virus etiology (OM not associated with a CLI and for which embedded virus assays were negative), and OM of unknown etiology (episode without a coexisting CLI and no virus sampling during the episode duration).
For consistency, the summary format for data presentation was average ± SD, which is used throughout.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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A total of 31 families were enrolled into the study in Year 1, and of these, 7 withdrew at some time before completing the study period. The reasons for withdrawal were that the study demands were overburdening to the family (n = 5) or the family relocated (n = 2). Consequently, the presented results focus on the data available for 60 children (3 black, 57 white) aged 4.4 ± 2.0 years (1.5–9.3 years) in the 24 families who completed the study and distributed as 2 children for 13 families, 3 children for 10 families, and 4 children in 1 family. Throughout the study period lasting from October 1 to April 30, a total of 513 nasal samples were collected and analyzed by PCR, yielding an average of 9.6 ± 2.5 (range: 2–16) samples per child (425 independent assays, see "Methods"); a total of 1513 bilateral pneumatic otoscopic examinations were done for an average of 25.2 ± 4.0 (range: 9–32) bilateral examinations per child, and the daily diaries were complete for 12296 child days, with an average of 205 ± 6 (range: 197–213) completed days per child. These results indicate excellent compliance with the planned otoscopic examinations and other data collections that is attributable primarily to the "in-home" visits made by study personnel.
Two-thousand seventy-seven of all child days (17%) were assigned by algorithm to an illness day, and the average cold burden when defined by the percentage of observation days assigned to an illness day was 17 ± 15% (range: 0%–68%) per child. These days were linked as 199 CLIs with an average rate of 3.3 ± 2.0 (range: 0–7) CLIs per child. Nasal samples were not collected during the CLI in 71 cases (36%) because of the lack of free secretions in children who refused to have nasal washing done, unavailability of the family during the CLI period (vacations, holidays), or the failure of the mother to assign and/or report a cold to study personnel during times when a CLI was identified retrospectively by the algorithm. Of the 128 CLIs with secretion sampling (64%), a virus was identified in 94 (73%). For individual children, virus was detected during an average of 2.1 ± 1.3 (range: 0–5) of the sampled CLIs for an average detection rate of 77 ± 27%. The distribution of viruses and virus combinations temporally associated with a CLI is reported in Table 1. As expected, for those CLIs with identified viral etiologies, rhinovirus was associated with >50% of the episodes.
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Of the 297 independent assays for virus performed during non-CLI periods, 54 (18.2%) were PCR positive for a virus or virus combination. Species distributions for these viruses are reported in Table 1. For all subjects, the average percentage rate of positive virus recovery given a non-CLI period was 16 ± 17% (range: 0%–67%). The relative frequency distribution of the viruses and virus combinations is similar to that for the CLIs, with rhinovirus most frequently identified, followed by RSV and influenza, and then adenovirus, parainfluenza, and coronavirus. Two-hundred forty-three specimens recovered during the non-CLI periods yielded no virus (81.8%) and, for individual subjects, the average frequency of negative virus recovery in the absence of a CLI was 84 ± 17% (range: 33%–100%)
Overall, there were 93 diagnosed OM episodes (either unilateral or bilateral). The average number of episodes per subject was 1.6 ± 1.3 (range: 0–4). Table 2 shows the distribution of viruses associated with these episodes when a CLI was diagnosed and when virus was identified in the absence of a CLI. Sixty-five of all OM diagnoses (70%) were related to a concurrent CLI (irrespective of concurrent virus recovery). Of these, secretion samples were not obtained in 10 cases (15%) and, in an additional 12 cases (18%), a virus was not identified despite inclusive sampling. The relative ordering of viruses by frequency of recovery for OM was not different from that presented for CLIs (see Table 1).
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More interesting is the relative frequency of OM during non-CLI periods but when viruses were identified in the nasal sample. There, an additional 18 OM episodes (19%) were concurrent with a virus isolated from the nose and nasopharynx, 6 OM episodes (6%) were not associated with viruses despite sampling during the OM episode, and 4 OM episodes (4%) could not be assigned because secretion samples were not collected during the episode. As with CLIs, the majority of these OM episodes were associated with rhinoviruses and a minority with adenovirus and coronavirus, but influenza and RSV were conspicuously absent.
For this population, the OM complication rate given a CLI was estimated at 65 OM episodes per 199 CLIs or 33%. Of the 79 OM episodes with concurrent nasal samples, 1 virus was isolated in 61 (77.2%). Of the 94 CLIs with virus recovery, 43 (45.7%) were complicated by a new OM episode. Finally, a new OM episode was associated with 18 (33.3%) of the 54 times virus was recovered from a nasal sample in the absence of a CLI.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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The results present a set of temporal coincidences among virus isolations from the upper respiratory tract, CLI expression, and OM as an otologic complication that suggest a causal relationship. Past studies in human volunteers support this interpretation by showing that experimental infection of the nose and nasopharynx with rhinovirus, influenza A, or RSV provokes a variable expression of signs/symptoms of illness that qualifies as a CLI episode in approximately two thirds of those exposed and otologic complications including OM in some but not all subjects.15–17,19 Other analyses of those data showed that illness and otologic expressions are decoupled; for example, a CLI is not a prerequisite to otologic complications, and the 2 show differential responses to prechallenge antibody titer and antiviral therapy.18,20,21 Because upper respiratory viruses are recovered from the nose and nasopharynx of infants, children and adults in the absence of overt illness,22–25 these results suggested to us that the OM complication rate of a vURI when based on a concurrent CLI underestimates the true rate, and similarly, that the percentage of total OM episodes explained by a vURI is much higher than previously reported.
The species distribution of viruses during CLIs in this study is similar to that reported previously, with rhinoviruses accounting for most (60%) illnesses, followed by influenza A and RSV at 10% and then adenovirus, coronavirus, and parainfluenza at 5% each.1,3,8 It is interesting that a similar relative frequency distribution for these viruses was documented for non-CLI periods with positive virus recovery. It is expected that this distribution reflects the proportional sampling over the entire "cold season" and that the frequency of virus recovery would be biased to a particular species by concentrated sampling during an endemic period.
Approximately 70% of OM episodes were temporally associated with a CLI, and where ascertainment was possible, there was no evidence of skewing in favor of specific viruses. Rather, the CLIs complicated by OM showed the same relative distribution of viruses as the source population from which they were drawn. This observation stands in contrast to some previous reports that specified a higher than anticipated frequency of OM episodes for a particular virus species.1,2,4–6 Because our result agrees well with more comprehensive, cross-seasonal surveys,3,8 we hypothesize that those reports were biased by factors that included dependence on signs/symptoms of illness for OM identification, the source population (eg, hospitalized children), and study season.
Finally, the study data reject our null hypotheses. Both hypotheses require that viral recovery during non-CLI periods not be associated with OM, a formulation that is inconsistent with our results. Rather, OM occurs as a complication of the presence of virus in the nose and/or nasopharynx irrespective of CLI presentation. Moreover, although the sample set is small, there does not seem to be a clear preferential ordering as to which viruses are associated with OM in the absence of illness. In that regard, our assessment of the presence or absence of a CLI was made by using an algorithm, and it is possible that the code set was insensitive to sign patterns specific to a given virus or to alternative sign presentations. To test this possibility, a 20-day period centered on each observation of a non–CLI-associated positive viral recovery was examined for concurrent signs. The results were consistent with a typically null set or a sporadic expression of signs that could not be clearly related to the virus (data not shown).
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CONCLUSIONS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Seventy percent of all OM episodes were attributable to a concurrent CLI episode, and, in this population of older children, OM was a complication in 33% of all CLI episodes. The primary etiologic agent associated with CLI and OM episodes was rhinovirus. Respiratory viruses were recovered during a large number of non-CLI periods, and these too were associated with the development of OM. More recently, other respiratory viruses such as metapneumovirus and bocavirus were identified as causing CLIs and precipitating OM, but their presence was not assayed in our study because the requisite techniques are not yet available in our laboratories.33,34 It is possible that these and other viruses were causative in those CLIs and OM episodes where our assay panel failed to identify a viral etiology. We conclude that most OM episodes are a complication of a vURI and that past age-adjusted estimates of OM as a vURI complication are biased downwardly by their dependence on CLI presentation.
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ACKNOWLEDGMENTS |
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This study was supported, in part, by National Institutes of Health grant DC005832.
We thank Kathleen Ashe for assisting with the virology assays; Margaretha L. Casselbrant, Harriette Wheatley, Kathy Tekely, and Ellen Reynolds for assisting with otoscopic examinations and sample procurement; and Julianne Banks and James T. Seroky for assisting with data entry.
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FOOTNOTES |
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Accepted Feb 13, 2007.
Address correspondence to Cuneyt M. Alper, MD, Children's Hospital of Pittsburgh, 3705 Fifth Ave at DeSoto St, Pittsburgh, PA 15213. E-mail: alperc{at}pitt.edu
The authors have indicated they have no financial relationships relevant to this article to disclose.
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