It has long been recognized that secondary bacterial infections are a primary complication of viral respiratory tract infection (106). In all areas of the world, upper respiratory tract (URT) infections and their complicating sequelae are the leading causes of acute infectious morbidity (138). Moreover, these situations of mixed microbial superinfection often result in increased morbidity or mortality over that caused by the primary viral or bacterial infection and are founded on synergistic processes in which specific diseases exist only if several microbes are present (133). Prior to having an understanding of the exact mechanisms that contribute to such microbial synergy, evidence of the etiology of viral-bacterial superinfections could be discerned from close examination of epidemiological data. These data often depict regular, and sometimes seasonal, patterns of peak periods of viral isolation (or detection of viral infection diagnostically) preceding those of bacterial disease. Otitis media (OM), or inflammation of the middle ear, is one of these complicating sequelae of URT infections and is indeed a major health care concern of childhood.
OM, both acute and chronic, is highly prevalent worldwide (34, 105, 189). The most recently available statistics indicate that 24.5 million physician's office visits were made for OM in the United States alone in 1990, representing a >200% increase over those reported in the 1980s (23, 153). OM is the most frequently diagnosed illness in children younger than 15 years of age and is the primary cause for emergency room visits (34). It is estimated that 83% of all children will experience at least one episode of acute OM (AOM) by 3 years of age and that more than 40% of children will experience three or more episodes of AOM by this age (195). Although only very rarely associated with mortality any longer, the morbidity associated with OM is significant. Hearing loss is the most common complication of OM (19, 104) with behavioral, educational, and language development delays being additional consequences of early-onset OM with effusion (OME) (105, 194). The socioeconomic impact of OM is also great; direct and indirect costs of diagnosing and managing OM exceed $5 billion annually in the United States alone (3, 33, 114, 189). To date, use of antibiotics, both therapeutic and prophylactic, has been largely relied upon for medical management of the spectrum of clinical entities known collectively as OM (189). Widespread use of antimicrobials for OM has met with controversy, however (189, 214), and the emergence of multiple-antibiotic resistant microorganisms is a sobering consequence of this well-established practice (46, 84, 139, 146). Surgical management of OM involves the insertion of tympanostomy tubes through the tympanic membrane (or eardrum) while a child is anesthetized. Although this procedure is commonplace (prevalence rates are ~13 per 1,000 children younger than 18 years of age) (24) and is highly effective in terms of relieving painful symptoms by draining the middle ear of accumulated fluids, it too has met with criticism because of the invasive nature of the procedure and the incumbent risks of putting a child under general anesthesia (21, 24, 153, 189).
Many environmental, anatomical, and other factors contribute to both the prevalence of middle ear infections in children and the chronicity or recurrent nature of OM. These factors include the immaturity of the pediatric immune system, existence of other ongoing infections, the anatomic positioning of the Eustachian tube in childhood, genetic predisposition, methods of feeding, smoking in the household, existence of allergies, and attendance at day care, among others (23, 32, 70). In addition, while children do mount an immune response both systemically as well as locally to the organism(s) present in their middle ears, due to the vast heterogeneity of the microorganisms that cause OM, this immune response does not confer protection against subsequent bouts of OM.
Whereas the multifactorial nature of middle ear infections is well acknowledged, it has only recently become fully appreciated that OM, both acute and chronic, is also a truly polymicrobial infection involving any of several URT viruses and one or more of three primary bacterial pathogens of the middle ear. This delayed understanding was partly due to difficulty in obtaining sequential samples from the middle ear for assay by culture and the fact that many middle ear fluids (or effusions) that were retrieved, in particular, from cases of chronic OM, were culture negative. Nevertheless, by the late 1960s there were a few reports that several laboratories were beginning to isolate viruses from the middle ear (parainfluenza virus type 2, respiratory syncytial virus [RSV], adenovirus [AV], rhinovirus, and coxsackievirus B4) either alone or in association with bacteria. In 1971, Gwaltney (85) hypothesized that OM etiology could be purely viral, purely bacterial, or the result of a mixed viral-bacterial infection. However, not enough evidence existed at the time to accurately state which constituted the primary infection of the middle ear or if there was indeed a single primary etiologic agent, nor was it easily determined how often each etiologic possibility occurred.
Infection and Etiologic Agents
Since then, an enormous amount of data has been amassed to indicate that, while one can indeed have a purely virus-induced inflammation of the middle ear or viral OM (118, 180), mixed bacterial and combined viral-bacterial infections of the tympanum are exceedingly common (36, 76, 107). With improved specimen collection and culture methodologies, even prior to the advent of powerful molecular detection methods, bacteria were cultured from 40 to 60% of middle ear fluids (MEFs) (76); virus alone could be isolated in approximately 6% of these fluids, and viruses plus bacteria could be cultured from approximately 17% of fluids recovered from the middle ear (5, 175). Moreover, at the time of OM diagnosis, more than 90% of patients were known to have signs of an URT infection, most likely viral in origin because 40 to 50% of these patients also had detectable virus in their nasopharynx (NP) (5).
A longitudinal study conducted by Henderson et al. (98) to determine the relative importance of viral respiratory tract infections or nasopharygeal colonization by Streptococcus pneumoniae or Haemophilus influenzae as factors influencing the occurrence of AOM with effusion (AOME) found a stronger association between infection with any of several viruses (RSV, influenza A or B virus, or AV) and AOME (average relative risk, 3.2) than between NP colonization with either of the two cited bacterial species and AOME (average relative risk, 1.5). Infection with RSV, influenza A or B virus, and AV also correlated with an increased risk of recurrent middle ear disease. In 1985, Sarkkinen et al. (180) demonstrated that, during a natural epidemic of RSV infection, there was a concomitant and significant increase in occurrence of AOM. The authors of these studies thereby suggested that prevention of selected OM-associated viral URT infections could reduce the incidence of middle ear infections in childhood, a concept that is still widely held today.
Thus, ample epidemiological evidence and data were generated by direct-culture methodologies to support the association of URT viruses with acute bacterial infection of the middle ear (6, 98, 119, 122, 176, 181, 182). Peak incidence of OM occurred in concert with peak periods of isolation of many URT viruses (98, 176) (Fig. 1). In addition to culture and epidemiological data, the involvement of RSV, rhinovirus, AV, parainfluenza virus, influenza virus, coronavirus, enterovirus, and others was clearly demonstrated in children with OM by multiple immunoassays (5, 38, 39, 118, 123). By the late 1980s, the crucial role of the respiratory tract viruses in the pathogenesis of bacterial OM was firmly established and has been the subject of several excellent reviews (76, 87, 89, 91, 92, 94, 176, 177) despite our having an incomplete understanding of the mechanisms involved. Exposure to respiratory tract viruses (primarily via attendance at a day-care facility or association with siblings who act as transmitters of viral pathogens) has become established as a significant risk factor and/or predictor for early-onset, frequent, or recurrent OM in multiple studies (126, 154). In fact, in a prospective study of 596 infants from birth to 6 months of age, Daly and colleagues found that exposure to URT viruses is indisputably the most important predictor for early AOM, outweighing all others with an associated relative risk of 7.5 (52).
Whereas evidence has accumulated over time to demonstrate that multiple URT viruses can be involved in the pathogenesis of OM, only three primary bacterial agents are commonly associated with infections of the middle ear. These are the gram-positive microorganism S. pneumoniae and the two gram-negative microorganisms, nontypeable H. influenzae (NTHI) and Moraxella catarrhalis. While the distinctions sometimes blur, S. pneumoniae, or the pneumococcus, is most commonly associated with highly symptomatic acute OM (or AOM), whereas NTHI and M. catarrhalis are more commonly associated with cases of OM that are less symptomatic (or "silent") but of longer duration (i.e., chronic OM [COM] or OME). Regardless of the relative degree of symptoms associated with a case of OM, the presence of microorganisms and fluid in the middle ear space compromises the functions of the ear, both hearing and balance.
Because of growing acceptance of the increased role of both viruses and bacteria in OM, despite the fact that by traditional culture methods approximately 30% of MEFs collected from children with AOM and 30 to 50% of MEFs from cases of OME were considered sterile (76), much effort was made throughout the 1990s to develop improved detection methods to better understand the microbial etiology of OM. The advent of more sophisticated, specific, and sensitive assays for the detection of viral and bacterial DNA and/or RNA lent further support to the role of URT viruses as the predisposing agents or copathogens of bacterial OM. One of the first reports of the detection of genomic sequences of viruses in effusions collected from children with OME was published by Okamoto et al. (152). These investigators demonstrated that RSV sequences could be amplified in 62% of middle ear effusions assayed during and/or after a natural outbreak of RSV in the community.
Many laboratories subsequently developed and reported the use of molecular assays to detect one or more viral pathogens (151, 161, 184), a single bacterial species (102, 201, 208), or mixed bacterial infections (99, 100, 167, 168) of the middle ear or their presence in NP secretions. The development and use of OM pathogen-specific multiplex assays to detect mixed viral-bacterial infections of the middle ear followed (18, 129). All of these assays have generated data that confirmed the commonality of mixed infections as the cause of OM, both acute and chronic, in animal models as well as in a clinical setting. Whereas by culture and immunodetection methods, viral involvement in OM has been reported to be between 30 and 50%, currently, by the use of PCR-based detection methods, the presence of at least one virus is detected in approximately 75% of children at the time of diagnosis of AOM, including positive viral detection in 48% of MEFs and in 62% of NP aspirates (162). Since bacterial superinfection of the middle ear, and the resultant effusion that develops, typically follow viral compromise, even this increase in reporting likely underrepresents viral involvement in OM.
Several rodent hosts (gerbil, rat, mouse, guinea pig, and chinchilla) have been used to develop models of single pathogen-induced OM through the years, and each has its own inherent strengths and limitations (54, 58). However, the first laboratory to establish an animal model of OM based on a mixed microbial infection of the middle ear was the Giebink laboratory, which clearly demonstrated in 1980 that the incidence of culture-positive pneumococcal OM was significantly increased if the chinchilla host was coinoculated intranasally (i.n.) with any of several isolates of influenza A virus (78) (Fig. 2). Whereas only 4% of chinchillas (4% of ears) inoculated with influenza A virus alone or 21% of animals (13% of ears) inoculated with S. pneumoniae alone developed OM, this incidence was increased to 67% (52% of ears) when the animals were dually challenged.
Following the example of Giebink, we attempted to identify an appropriate viral copartner for NTHI to better understand the pathogenesis of OM due to this gram-negative organism. An improved model was needed because NTHI alone was incapable of consistently inducing culture-positive OM from NP-colonized chinchillas when inoculated as the sole pathogen (187, 192). Moreover, existing transbullar models wherein NTHI was injected directly into the chinchilla middle ear, while resulting in severe OM in virtually all animals, did not represent the natural disease course in children. Attempts to partner the same strain of influenza A virus used by the Giebink laboratory (influenza A/Alaska/6/77) with NTHI were unsuccessful; coinoculated animals had no greater incidence of OM than those receiving NTHI alone (author's unpublished observations). However, we later showed that a clinical isolate of AV (serotype 1) did indeed predispose juvenile chinchillas to culture-positive middle ear disease when partnered with NTHI (192). Animals that received NTHI by i.n. delivery 7 days after challenge with AV developed OM of greater severity than did cohorts that received either NTHI alone, AV alone, NTHI and AV at the same time, or NTHI 7 days prior to receiving AV. This dual challenge model resulted in the greatest incidence of culture-positive OM, the most prolonged presence of NTHI in the NP and middle ears, and the most severe damage to the middle ear mucosa and altered Eustachian tube function.
This study was followed by an investigation of the kinetics of development of this mixed infection, again in the chinchilla host (143). By using snap-frozen sections of Eustachian tube and middle ear mucosa to assay for adherent bacteria via fluorescent and transmission electron microscopy, we found that NTHI gradually ascended the Eustachian tube, in a retrograde fashion, from an NP colonization site to the middle ear cavity by adhering to mucus on the floor of the Eustachian tube (Fig. 3a and b). NTHI reaches the tympanum approximately 7 to 10 days after being introduced i.n. to AV-compromised chinchillas. Our ability to detect NTHI adhering to middle ear mucosa very close to the tympanic orifice of the Eustachian tube coincided with the onset of signs of OM in these animals (192) (Fig. 3c).
Thus, it became evident that the partnering of virus with bacterium and the timing of this interaction in the chinchilla host represented a highly specific interrelationship. Moreover, OM pathogenesis was clearly not predicated solely on general viral compromise of the uppermost airway. Whereas influenza A virus did indeed predispose to pneumococcal OM and AV similarly sets the stage for invasion of the middle ear by NTHI, we found, as stated above, that influenza A virus does not predispose the chinchilla host to NTHI-induced OM. Nor does AV predispose to either M. catarrhalis-induced OM (17) or that caused by S. pneumoniae (197). Recent data from my laboratory (unpublished data) suggest that, in M. catarrhalis-induced OM, the etiology of the infection may require more than a single viral or bacterial copathogen and thus, the situation is more complex than what we currently believe to be the case for at least some infections of the middle ear due to S. pneumoniae or NTHI. Similar specificity of virus/bacterium synergy appears to be maintained in adults and children. The oropharynges of 15% of adults with experimental influenza A virus infection became heavily colonized with S. pneumoniae 6 days after challenge (209), whereas isolation rates for other middle ear pathogens were not affected by infection with this virus. In children, S. pneumoniae is cultured significantly more often from MEFs that contain influenza A virus than those that are culture positive for either RSV or parainfluenza virus (97).
Although other rodents have not yet been used to model OM of mixed microbial etiology as has been done with the chinchilla, all the rodent models cited earlier have contributed a tremendous amount of information regarding the mechanisms by which the URT viruses predispose to bacterial invasion of the middle ear (reviewed below). Thereby, the importance of developing and utilizing relevant animal models cannot be overstated even in light of their inherent limitations. In fact, contributing greatly to the relative lack of progress made to date in our understanding of the pathogenesis of M. catarrhalis-induced OM, and consequently methods to prevent it, is the lack of a good animal model of middle ear disease induced by this pathogen. Models established to date for M. catarrhalis in mice, chinchillas, and rats lack many of the hallmark signs of an ongoing disease process and all demonstrate unequivocally the rapid clearance of M. catarrhalis from the airway and/or middle ears of these hosts (42, 58, 103, 204, 212).
Mechanisms of Pathogenesis
Multiple mechanisms have been identified that serve as contributing factors in the synergistic relationship between the URT viruses and the primary bacterial pathogens of OM, and while each of these is a highly specific effect, all fall within the general category of compromise of airway defenses. In most cases, it is highly likely that more than one of these mechanisms is operational at any given time and quite possibly varies depending on the time point within the multifactorial disease course of OM (12). Again, we have a better understanding of these processes as they relate to S. pneumoniae and NTHI to date than we do for M. catarrhalis, due in part to the absence of a useful model to help us define the pathogenic mechanisms of infection of the middle ear with this latter group of microorganisms. Some of these mechanisms are reviewed below.
Viral Effects on Bacterial Adherence and/or Colonization
In the late 70s, a hypothesis was put forth that one explanation for the commonly observed association between virus infection (specifically, influenza virus) and subsequent bacterial superinfection could be that cells infected with certain viruses might be more permissive to adherence of bacteria, which in turn could promote bacterial colonization and ultimately lead to infection and disease (179). Testing of this hypothesis was directly relevant to the study of the pathogenesis of OM when several investigators demonstrated that some of the URT viruses did indeed augment adherence by the bacterial pathogens of OM. Influenza A virus increases the adherence of type I S. pneumoniae to mouse tracheal epithelial cells (163) but not that of NTHI to chinchilla tracheal epithelium in an organ culture system (14). Hakansson et al. (86) showed in vitro that infection with AV types 1, 2, 3, and 5 significantly enhances the binding of adherent strains of S. pneumoniae that had been isolated from the NP of children with frequent episodes of AOM, to human lung epithelial cells. Their data suggest that AV upregulates the expression of receptors for S. pneumoniae on the surface of these respiratory epithelial cells. Jiang and colleagues (108) later demonstrated that RSV infection of A549 cells similarly significantly enhances attachment of NTHI that express outer membrane protein P5-homologous fimbriae but not the attachment of an isogenic mutant that does not express this adhesin (Fig. 4). This increase in adherence does not correlate with the relative amount of RSV antigen expressed by these human lung epithelial cells. However, UV-irradiated supernatants collected from RSV-infected cells also significantly enhances the attachment of fimbriated NTHI to A549 cells suggesting the presence of a preformed soluble precursor in these supernatants that enhances expression of a receptor for this NTHI adhesin.
With use of flow cytometry to detect bacteria adhering to influenza A virus-infected Hep-2 cells, El Ahmer et al. (61) recently showed that viral infection significantly increased adherence by all three groups of microorganisms most commonly associated with both AOM and COM. In their studies, 8 of 8 M. catarrhalis isolates, 5 of 5 respiratory tract isolates of S. pneumoniae, and 2 of 2 NTHI isolates showed significantly increased adherence to this particular target cell. By using a series of monoclonal antibodies to assay for potential changes in expression of cell surface antigens that could act as receptors for bacterial adherence, these investigators also found that infection of Hep-2 cells with influenza A virus resulted in a significant increase in binding of antibodies specific for both CD14 and CD18 but not Lewisb, Lewisx, or H type 2 markers. CD14 and CD18 have been shown to serve as a receptor site for adherence of several gramnegative bacteria, and thus virus-induced up-regulation of host cell surface antigens that can act as bacterial receptor sites appears to be a common theme in the pathogenesis of OM and other diseases of the respiratory tract.
Since the origin of bacterial invaders of the middle ear are those microorganisms that colonize the NP, the finding that viral infection promotes bacterial adherence to airway epithelial cells has enormous implications for the disease course of OM. Early studies by Fainstein et al. (69) had shown that more type I pneumococci and H. influenzae adhere to pharyngeal cells collected from human volunteers experimentally infected with influenza A/USSR/77 after infection than to pharyngeal cells collected prior to challenge. Surprisingly, individuals with natural URT infection did not similarly demonstrate this enhanced adherence. More recently, Patel and colleagues (156) showed that colonization of respiratory tract epithelium by NTHI is increased upon exposure of cotton rats to RSV. Colonization of the NP by NTHI is significantly greater 4 days after RSV infection than in RSV-negative controls. However, thereafter, NTHI is cleared more rapidly from the NP of RSV-infected animals than from controls, and adherence of NTHI to cells collected from RSV-infected cotton rats at the time of maximal virus replication is not different than in these control animals. These data suggested that, whereas infection with RSV does temporarily induce significantly augmented colonization of the NP in the host, the exact mechanism(s) by which this occurs is not yet clear. With use of a chinchilla host, influenza A virus (but not AV) has similarly been found to enhance nasopharyngeal colonization by S. pneumoniae, and particularly by a strain with an opaque versus a transparent phenotype (197, 198). Hirano et al. (101) reported that influenza A virus infection of mice results in increased colonization by both NTHI and S. pneumoniae; however, the increase in recovery of NTHI was found to be significant only on day 5 after challenge when compared with controls. Conversely, a significant increase occurred in recovery of S. pneumoniae in nasal lavage fluids on days 5, 9, and 14 after influenza A virus challenge.
The positive correlation between viral URT infection and NP colonization has also been shown in human challenge models (209) wherein influenza A virus infection promotes colonization with S. pneumoniae. Moreover, NP colonization alone has been positively correlated with an increased incidence of OM in children (66, 67). In fact, even in the absence of a viral URT infection, children are colonized with the organisms that induce OM very soon after birth. Faden and colleagues (63) have shown that, at 6 months of age, 26% of infants are already colonized with M. catarrhalis, 24% with S. pneumoniae, and 9% with NTHI. By 1 year of age, these percentages increase to 72, 54, and 33%, respectively. These investigators have also shown that early colonization is associated with early initial episodes of AOM and that colonization with either S. pneumoniae or H. influenza in the first year of life increases the risk of becoming otitis prone fourfold compared with the absence of colonization with these microorganisms (Fig. 5). In addition, there is a direct relationship between the frequency of colonization and the frequency of AOM.
Despite their prevalence as colonizers of the pediatric NP, all three bacterial species associated with AOM and COM typically behave as benign commensals (or normal flora) and as such are largely ignored by the host. However, when children are also infected with a URT virus and thus experiencing compromise of their Eustachian tubes, these organisms often act as opportunistic pathogens. During URT viral infection, because of the temporary inability of the Eustachian tube to prevent their ascent (see the next section), bacterial commensals of the NP can gain access to the middle ear and begin to multiply, sometimes causing severe pain and/or long-term infection of the middle ear. These infections of the middle ear, and particularly those that occur very early in life, can induce pathological changes in the middle ear that set the stage for subsequent recurrent or chronic OM.
Viral Compromise of Eustachian Tube Function
Virus-induced damage to the mucosal epithelium lining the uppermost airway and the effect of this pathology on airway function, particularly that of the Eustachian tube, has also been shown to contribute significantly to bacterial superinfection of the middle ear (23, 142). In children, the Eustachian tube is shorter and more horizontal in orientation than in adults. The immature Eustachian tube is also more compliant (or "floppy") and the mechanisms for active opening of the tubal lumen, largely via the action of the tensor veli palatini muscle, are not yet fully effective (Fig. 6). These attributes make the pediatric Eustachian tube naturally more susceptible to invasion by microbes from the NP; however, this susceptibility is significantly enhanced during times of viral compromise.
The effect of influenza A virus on the chinchilla Eustachian tube after i.n. inoculation was investigated by Giebink et al. (80) in an attempt to understand the mechanisms underlying the negative pressure recorded in the middle ears of these animals when infected with various strains of this virus. Inflammation of the tympanic membrane and an underpressured state of the middle ear mirrors both epithelial damage in the Eustachian tube and the accumulation of mucus and cellular debris in the tubal lumen (Fig. 7). Epithelial damage was found to be greatest in the proximal two-thirds of the Eustachian tube, whereas goblet cell metaplasia and increased secretory activity was greatest in the distal, tympanic one-third of the tube after i.n. challenge with influenza A/Alaska/6/77 virus. These data thus provided the first morphologic correlate for the development of negative middle ear pressure and contributed to our understanding of the basis for purulent OM that occurred during viral respiratory tract infection. Ohashi et al. (150) later reported similar findings in a guinea pig model following intratympanic inoculation of influenza A virus. However, whereas the character of the histopathology noted was highly reminiscent of that shown in the chinchilla, in their study, virus-induced damage was greatest more proximal to the tympanic bulla because of the direct challenge of the middle ear in this latter model.
The overall finding that URT viruses compromise Eustachian tube function in guinea pigs and chinchillas correlates well with virus-induced changes that occur in human volunteers challenged with rhinovirus or influenza A virus. Rhinovirus-induced effects on middle ear pressure status and nasal patency was measured in sequestered human volunteers challenged i.n. with this virus (26, 137). Abnormal middle ear pressures, decreased nasal patency, and depressed tubal function occur in 50% or more of the ears of subjects that developed a clinical illness (or "cold") due to rhinovirus. In another study, volunteers were inoculated with influenza A virus. More than 80% developed Eustachian tube dysfunction and middle ear underpressures of less than −100 mm H2O on days 4 and 5 after challenge (59). Five of 21 subjects with low prechallenge antibody titers to influenza A virus also developed OME. These data thereby collectively support a causal relationship between viral URT infection, Eustachian tube obstruction, abnormal middle ear pressure, and OM.
The degree to which a particular virus compromises the airway, particularly the Eustachian tube, has a tremendous influence on whether or not OM is induced and, if so, how severe the disease course is. Giebink and Wright (81) clearly demonstrated this influence when they showed that different strains of influenza A virus had markedly altered levels of virulence in the chinchilla host when the outcome measures were: the development of a negative middle ear pressure, neutrophil dysfunction, and increased susceptibility to pneumococcal OM. These data thus supported epidemiological evidence that had shown a striking difference in prevalence of AOM associated with different influenza A outbreaks. Whereas 41% of children with A/Texas influenza had associated AOM, only 10% of those with A/USSR or 18% of those who were not infected with either virus strain had OM.
These data are in keeping with the general principle that, although nearly all respiratory tract viruses can predispose to bacterial OM, there does seem to be both intra- and inter-strain variability in their relative ability to do so. Uhari et al. (202) prospectively studied 658 children to analyze the etiology of respiratory tract infection with and without associated AOM. Of 197 children with AOM, the only virus that was more commonly isolated in these patients was RSV. While these investigators monitored children who were admitted to the hospital, and thus they studied a patient population that was more severely ill than the typical child with OM, many laboratories have reported the stronger association of RSV with AOM than many other URT viruses (92, 97, 98, 118, 119, 122, 162, 176, 180). Thus, those viral URT infections that are associated with more severe compromise of the uppermost airway appear to be those that enhance susceptibility to bacterial OM.
In animal models, the time between initial viral infection (or challenge) and bacterial invasion of the middle ear is approximately 9 days for the pneumococcus after influenza A virus inoculation (78) and 7 to 10 days for NTHI after AV inoculation (143). This time is similar to the interval between onset of symptoms of upper respiratory infection and AOM as shown epidemiologically. In a study of 204 cases of AOM that occurred in association with 412 episodes of URT infection, 75% of cases occurred during the first week after onset of symptoms and 85% occurred during the first 9 days (93). Koivunen et al. (121) recently examined 250 episodes of AOM in children and similarly found that 63% of AOM complicating URT infection occurred in the first week and 89% occurred by the end of the second week after onset of URT infection (Fig. 8). The greatest incidence of AOM was observed 2 to 5 days after the onset of respiratory symptoms. This interval between viral infection and bacterial superinfection of the ears was directly documented by Buchman et al. (27), who demonstrated the invasion of the middle ear of one subject by S. pneumoniae by PCR amplification of pneumococcal DNA from a middle ear effusion recovered 5 days after challenge with influenza A virus. These observations thereby fit well with the physician- and parent-described impression that "… a child gets a cold and a week later has an ear infection" (76).
Viral Effects on Antibiotic Efficacy
Both Arola et al. (7) and Chonmaitree et al. (37) first suggested the association between viruses and antibiotic treatment failure in patients with OM. Because OM is generally considered to be a "bacterial disease," antibiotic therapy was at one time recommended for all patients with AOM. In 4 to 18% of these patients, however, the signs of infection persist despite the fact that in approximately 81% of these cases the microbes isolated from the middle ears are susceptible to the prescribed antimicrobial (7). Both groups observed that patients who were unresponsive to treatment with antibiotics also tended to have positive viral cultures of the middle ear (Table 1). In a later prospective study of 271 infants and children with AOM, Chonmaitree et al. (38) found evidence of viral infection in 46% of these patients, 76% of whom also had bacteria present in their MEFs. More of the patients with this combined bacterial-viral infection (51%) had persistent OM 3 to 12 days after institution of antibiotic therapy than either those with bacterial OM (35%) or patients with OM due to a viral pathogen alone (19%). Other prospective studies corroborated the linkage between viral involvement in OM and antibiotic treatment failure (88, 157, 190). Collectively, these data suggested that the presence of viruses in the MEFs might be interfering either directly or indirectly with the clinical response to antibiotics. The exact mechanisms by which concomitant viral infection might be a determinant of treatment outcome in some AOM episodes (despite the susceptibility of the bacterial pathogen cultured from the middle ear to the antibiotic prescribed) thus became the subject of several investigations.
One explanation for the commonality of treatment failure in cases of viral-bacterial OM may lie in the combined effect of viral and bacterial invasion of the middle ear on penetration of antimicrobials from the bloodstream into the tympanum. With a diffusion model developed in chinchillas, Jossart et al. (111) compared middle ear elimination rates for three antimicrobials in four groups of animals: (i) controls that were not challenged with any microbe, (ii) those inoculated with influenza A virus alone i.n., (iii) those infected with both influenza A virus and S. pneumoniae by direct inoculation into the middle ear, and (iv) those inoculated directly into the middle ear with S. pneumoniae alone. After infection was established, a solution of amoxicillin, sulfamethoxazole, and trimethoprim was first instilled into the middle ear, then removed 4 h later. The authors of this study present the argument that, because passive diffusion occurs in both directions, and there is no evidence of an active transport mechanism for antibiotics into or from the environment of the middle ear, the model assumes that the rate of antibiotic elimination from the MEFs equals the rate of antibiotic penetration from the bloodstream, through the middle ear mucosa into the middle ear. Thereby, the rate constant of elimination and half-life of the antimicrobials were calculated from drug concentrations at the stated time points, and these values were used as a marker for antimicrobial penetration into the middle ear.
These investigators found that S. pneumoniae infection alone significantly shortened the middle ear elimination half-life of all three antimicrobials compared with the control group; the combined influenza A virus plus pneumococcus infection significantly lengthened the half-life of all three antimicrobials compared with the pneumococcal infection alone; and influenza A virus itself resulted in the longest half-lives for all three antimicrobials. Thus, the decreased penetration of antimicrobials into the middle ear that they theorized would occur in infections of combined viral-bacterial etiology, compared with those due to bacteria alone, supported the clinical observation that patients with infections of mixed etiology may have decreased middle ear antimicrobial concentrations which in turn leads to treatment failure.
A subsequent study of 34 children with AOM was conducted by Canafax et al. (29) to prospectively evaluate whether viral coinfection in AOM reduced antibacterial efficacy of antibiotics by determining the penetration and pharmacokinetics of amoxicillin in either bacterial or combined bacterial and viral AOM. Middle ear fluids, nasal wash fluids, and serum were collected from all 34 children at selected times between 0.5 and 4 h after oral dosing (40 mg/kg/day) and geometric mean amoxicillin concentrations were determined. Lowest values were associated with children infected with the virus only (2.7 μg/ml in MEFs) and these values were similar to those obtained in children that yielded culture-negative effusions (2.9 μg/ml in MEFs). Geometric mean amoxicillin concentrations were higher in children with combined bacterial and viral infection (4.1 μg/ml in MEFs) and were highest in those with bacterial-only infections of the middle ear (5.7 μg/ml in MEFs). Thus, there was indeed lesser penetration of amoxicillin from serum into MEFs when virus was also present in the middle ear, providing additional evidence for a mechanism whereby a higher incidence of antibiotic treatment failure occurs in these individuals.
A potential link between treatment failure, the presence of virus in the middle ear, and levels of specific inflammatory mediators in MEFs was investigated by Chonmaitree et al. (41), who measured levels of interleukin-8 (IL-8), a polymorphonuclear neutrophil (PMN) chemotactic cytokine, and leukotriene B4 (LTB4), a potent inflammatory product of PMNs, in 271 MEFs collected from 196 children with AOM. Forty-two percent of these children had evidence of respiratory viral infection as well. Levels of both LTB4 and IL-8 were significantly higher at the time of diagnosis in children with either bacterial AOM or mixed bacterial-viral infection than levels in MEFs from culture-negative children. No virus-related effect was observed for IL-8 when bacteria were absent; however, these levels were significantly higher in MEFs that contained both bacteria and viruses than in all other groups. Moreover, in children who had both bacteria and virus isolated from one MEF but were virus-negative in the contralateral MEF, levels of both IL-8 and LTB4 were higher in the MEFs that contained both bacteria and virus in all but one case wherein the LTB4 level was greater in the MEF that was virus negative. Bacteriologic failure after 2 to 5 days of treatment with either of two antibiotic regimens was significantly associated with high LTB4 levels in initial MEFs, whereas recurrence of AOM within 1 month was associated with high IL-8 levels in these effusions. These findings suggest that both of these PMN-related inflammatory substances are produced during acute infections of the middle ear and may play an important role in delayed recovery and/or recurrence of disease.
Viral Effects on Host Immune Functions
Another topic of enormous interest as it relates to the pathogenesis of OM is the effect of URT viruses on host immune function. The global effect of viral infection on neutrophil (2) and alveolar macrophage (211) function has been appreciated for some time and transient peripheral PMN dysfunction has been reported in children with recurrent OM (79). Neutrophil dysfunction has also been linked with influenza A virus infection in a chinchilla model (1). Abramson et al. (1) reported significantly depressed chemotactic, chemiluminescent, and bactericidal activities 4 to 8 days after inoculation of virus when compared with controls. Although they did not directly test this, the authors suggested that the known increased susceptibility of chinchillas to pneumococcal OM after inoculation with influenza A virus (78) might be due, in part, to these impaired chemotactic and oxidative microbiocidal activities of the neutrophils.
Viral-mediated release of cytokines and inflammatory mediators in the respiratory tract is also likely involved in the pathogenesis of OM. Cytokine activity in nasopharyngeal secretions collected during the course of a primary RSV infection shows the local production of IL-6 and tumor necrosis factor alpha (TNF-α) in 100% and 67% of infants and children, respectively (136, 147). Similarly, experimental influenza A virus infection in 17 adult volunteers led to significantly increased levels of IL-6, but not IL-4, in lavage fluids of all 12 subjects who shed virus (75). Increased levels of this pleotropic cytokine were not found in the five subjects who did not shed virus. Noah et al. (136, 147) examined children in a day care setting during acute upper respiratory infection, including children with OM, and also found markedly elevated levels of IL-1β, IL-6, IL-8, and TNF-α in nasal lavage fluids that were not dependent on the specific virus isolated. In addition, nasal mucosal biopsies showed increased transcripts for IL-1β, IL-6, and IL-8 in the epithelial cells of seven of nine subjects, thus suggesting that epithelial cells are a source of these proinflammatory cytokines in the nasal cavity. In these children, the levels of all cytokines, but not TNF-α, decreased significantly within 2 to 4 weeks.
The enhancing effect of combined bacterial-viral infections on production of inflammatory mediators in the middle ear by the host has also been shown (40). Histamine levels were measured in 677 MEF samples collected from 248 children with AOM, of which 47% had a documented viral infection as well. Histamine content was found to be significantly higher in either bacteria-positive or virus-positive fluids versus samples that were negative by culture, and together, bacteria and viruses have an additive effect on histamine content in MEFs. Thus histamine production is induced by infection of the middle ear and most markedly in situations of mixed microbial infections. High levels of histamine, cytokines, and other inflammatory mediators in MEFs can lead to increased inflammation in the middle ear and this enhancement of the host's immune response is, in essence, a double-edged sword. While an inflammatory response is essential for microbial clearance, it is also believed to contribute to both the pathogenesis and prolongation of OM via disease-associated tissue destruction and interference with penetration of antimicrobials into the site, among other mechanisms (94). For example, histamine also causes impaired ciliary activity and induces mucosal swelling in the tubotympanum thus prolonging mucociliary clearance time from the tympanic cavity. In fact, because of these known compromising effects, intratympanic instillation of histamine is now used to predispose to pneumococcal disease in a recently developed rat model of OM (207).
Viral Effects on Rheological Properties of Mucus and Mucociliary Transport
Many URT viruses, including those commonly associated with OM, are known to induce ciliary ultrastructural abnormalities or selective destruction of ciliated cells in airway epithelium (31, 163). Both of these mechanisms can compromise mucociliary clearance mechanisms throughout the respiratory tract, including in the nasal cavity of children with acute viral infection (31). Park et al. (155) studied influenza A virus-induced histopathology in chinchillas, including suppression of ciliary activity of the mucosa lining the Eustachian tube, and the subsequent impaired ability of this organ to move a small bolus of fluid from an inferior aspect of the middle ear cavity to the back of the throat where it can be swallowed. In this study, and in another by Chung et al. (43), influenza A virus induced pronounced damage to cilia and ciliated cells in addition to causing an extensive infiltration of PMNs into the subepithelial space. There were minimal changes to goblet cells.
Concurrent with these morphological changes was compromise of chinchilla Eustachian tube functions with maximal effects observed approximately 7 to 14 days after challenge (155). Ciliary beat frequency decreased from a normal rate of 19.2 ± 0.1 to 15.9 ± 0.3 Hz at the tympanic portion and 18.3 ± 0.1 Hz at the pharyngeal portion of the Eustachian tube on day 7 after transbullar inoculation (Fig. 9A). Interestingly, 10 days after i.n. inoculation of influenza A virus, ciliary beat frequency values were significantly decreased at both the tympanic and pharyngeal portions of the Eustachian tube (14.6 ± 2.6 or 14.8 ± 2.3 Hz, respectively) (Fig. 9B). This slowing of ciliary beating, combined with the discoordinated activity among ciliated cells that also occurs as a result of viral infection, leads to compromised clearance function. Thus, the ability of the mucociliary escalator of the Eustachian tube to transport fluid out of the middle ear space was similarly maximally diminished on days 7 to 14 after transbullar challenge. Transport times increased from a normal time of approximately 150 s to a situation in which there was no appearance of dye at the pharyngeal orifice of the Eustachian tube even 15 min after its instillation into the middle ear (Table 2). Ohashi et al. (150) reported similar decreased ciliary activity and increased time to clearance (from 68.3 ± 7.5 s to a maximum of 155 ± 19 s on day 14) in guinea pigs inoculated with influenza A virus intratympanically.
AV also compromises Eustachian tube function in chinchillas (13); however, for this virus, maximal compromise occurs 10 to 21 days after i.n. inoculation or 7 to 14 days after transbullar inoculation of this host. Subepithelial hemorrhage and edema, intense PMN infiltration, and clumping and shortening or loss of cilia were observed in addition to focal necrosis and sloughing of epithelium to reveal the basal cell layer in sections of Eustachian tube recovered from these animals. The presence of intranuclear inclusions and marked hyperplasia of goblet cells were consistent with AV infection.
Whereas the exact nature of the induced histopathology in both the Eustachian tube and middle ear is indeed virus specific in these models of influenza A virus or AV infection, the net effect in all cases is a severe compromise of Eustachian tube function. This compromise results in a temporal loss of ability of this organ to function as a primary defense mechanism of the middle ear, keeping bacteria and/or additional viruses from invading the middle ear from the NP. At the point of maximal compromise of the epithelium lining the Eustachian tube, as measured by reduced ciliary beat frequency and significantly ameliorated ability to move a bolus of dye from the middle ear to the pharynx, the ascension of the Eustachian tube by bacteria colonizing the NP can be detected (192). Return to normal epithelial organization, ciliary morphology, and mucociliary clearance function after virus-induced damage can take 28 days after experimental influenza A virus infection in guinea pigs (150) or chinchillas (43, 155) (Fig. 10), approximately 35 days in chinchilla models of AV infection (43, 192) (Fig. 11) and 2 to 10 weeks in children (31). Thus, the middle ear is most susceptible to ascending bacterial infection during these periods of recovery after URT infection. Given the variety of viruses a child is exposed to annually and the time it takes for the middle ear to recover after each bout of viral URT infection, one can easily imagine the scenarios that lead to AOM, recurrent OM, and chronic OME.
The specificity of the interrelationship between the viral and bacterial pathogens of OM was discussed above. One possible explanation for the specificity of this synergy resides in the fact that several viruses are known to have a distinct effect on the character of mucus secreted into the uppermost airway. For example, AV leads to both an increase in relative amount and viscosity of NP secretions in chinchillas challenged with a serotype 1 isolate (13); however, the biochemistry of these secretions is not altered (192). By using a panel of 15 lectins with a broad range of specificity for labeling of cell surface carbohydrates, we found that there was no marked change in either labeling intensity or distribution of label between AV-infected chinchillas and control animals. The goblet cell hyperplasia noted to occur in the mucosa that lines the Eustachian tube is a hallmark of AV infection in chinchillas and provides a possible mechanism for the noted hypersecretion of mucus in these animals (Fig. 12). This hypersecretion phenomenon combined with an inability to adequately hydrate this increased amount of "normal" mucus likely accounts for the changes in the rheological properties of airway secretions that we have reported. This situation seems to favor ascension of the Eustachian tube and initiation of OM due to NTHI. In fact, NTHI adheres to mucus within the Eustachian tube lumen of AV-infected chinchillas rather than adhering to any particular cell type in this site (144) (Color Plate 2 [see color insert]).
Influenza A virus, on the other hand, also leads to an increased production of respiratory secretions in the first week after exposure of human volunteers (60). However, these secretions are not more viscous than normal and, unlike the situation with AV, there is a concomitant change in the biochemical character of the mucus blanket and epithelial cell surface carbohydrates attributed to the action of viral neuraminidase. Hirano et al. (101) inoculated mice with influenza A virus and examined their NP mucosa for changes in labeling patterns using a battery of lectins. Staining of the mucus blanket and epithelial cell surface with lectins (PNA, succWGA, and BSL-II) specific for either GlcNAc or Gal residues was significantly increased compared with controls, whereas labeling with lectins (WGA or MAA) with specificity for terminal sialic acid residues was only moderately enhanced in virus-infected animals and only on days 5 and 9 after virus inoculation. Because terminal glycosylation sequences of epithelial cell surface and mucus carbohydrates mediate adherence by microorganisms, the effects of viral infection on these structures has important implications for the pathogenesis of OM.
Effects of Bacteria and Their Products on Airway Physiology and Function
While the URT viruses have been studied more extensively in terms of their synergistic relationship with bacteria in causing OM, the influence of the exact bacterial pathogen (or pathogens) present in MEFs on the course and severity of OM has also been a topic of some investigation. Bacteria, like viruses, elicit the production of inflammatory mediators in the middle ear, and several investigators (8, 9, 44, 55, 95, 109, 110, 112, 140, 145, 149, 183, 193, 196, 213, 215) have reported the elicitation of many proinflammatory cytokines, growth factors, and products of complement activation in MEFs in response to invasion of the middle ear by the bacterial pathogens of OM. However, to date, the inflammatory mediators elicited by each of these microorganisms and the effect they have on subsequent superinfection have not been fully elucidated. Nevertheless, a few studies have looked at the effect of bacterial products, particularly endotoxin (lipooligosaccharide [LOS] or lipopolysaccharide [LPS]) or extracellular enzymes, on the epithelium that lines the uppermost respiratory tract.
Ohashi et al. (149) found that LPS isolated from Klebsiella pneumoniae induces a marked decrease in ciliary beat frequency in guinea pig tubotympanal mucosa. Likewise, Bakaletz et al. (11) showed that either LOS isolated from Salmonella enterica serovar typhimurium or whole formalin-fixed NTHI, but not formalin- fixed S. pneumoniae, had a significant suppressive effect on the ability of the chinchilla Eustachian tube to transport fluid. Injection of purified LOS or a killed gram-negative (and thus endotoxin-containing) organism (NTHI) into the middle ear cavity results in an early production of an MEF, capillary leakage, and slowing of mucociliary transport.
Bacterial enzymes also likely play a role in the pathogenesis of OM. Neuraminidase purified from S. pneumoniae is known to alter chinchilla middle ear mucosa by removing sialic acid residues and exposing galactose residues of cell surface carbohydrates expressed on mucosal epithelial cells (128). Linder et al. (130, 131) further investigated the effects of pneumococcal extracellular enzymes, particularly neuraminidase, on the biochemistry of the tubotympanum-using chinchilla models. They found that inoculation with S. pneumoniae either via a transbullar or i.n. route induced a change in lectin-labeling pattern from that obtained with use of naive or control tissues, which demonstrates, like Doyle et al. (128), that terminal sialic acid residues had been removed and N-acetylglucosamine residues had been exposed. There was a marked increase in labeling with lectins (PNA, succWGA, BSL II or ECL) specific for either GlcNAc or Gal, whereas there was a decrease in labeling with lectins (SNA and WGA) specific for terminal sialic acid residues. Thus the pneumococcus, through the action of one of its extracellular enzymes, can induce the exposure of its own host cell receptor site, specifically Galβ1-4Glc-NAcβ1-3Galβ. As discussed above, influenza A virus neuraminidase affects lectin labeling patterns in the uppermost airway in a similar manner which also likely contributes significantly to the specific association between this URT virus and pneumococcal OM (101).
Some evidence shows that the presence of one bacterial species in an MEF may alter the survival of others in these fluids (188). Viable M. catarrhalis appears to prolong the survival of both S. pneumoniae and H. influenzae when coincubated in sterile mucoid effusions collected from patients with ongoing secretory OM, whereas nonviable M. catarrhalis enhanced the growth of S. pneumoniae only. Conversely, both the pneumococcus and H. influenzae suppressed the growth of M. catarrhalis when coincubated in these mucoid MEFs. Although the exact mechanism for these variable effects on survivability are not known, the authors suggested that these phenomena are also likely in play in the middle ear during natural disease. Thus, the course of disease development and resolution that occurs in the middle ear can be highly variable and largely depends on which microbe, or combination of microbes, initiates the infection as well as those that contribute to the superinfection.
Biofilms and OM
Despite the culture-negative status of 40 to 60% of MEFs, PCR-based assays consistently suggested a much higher incidence of bacterial involvement in OM. Due to the many-fold greater sensitivity of PCR, however, the early PCR-generated data were viewed somewhat skeptically and there were concerns as to whether these data were more reflective of false-positive results than an actual increased involvement of bacteria in COM or OME. Thereby, to determine whether the PCR-generated data were truly indicative of the existence of viable but not culturable bacteria in the middle ear or merely due to the persistence of residual "fossilized" DNA in the absence of viable bacteria, Post et al. (165) and later Aul and colleagues (10) designed a series of studies to address these concerns specifically and systematically. They found that purified DNA and DNA from intact but nonviable (heat-killed) bacteria do not persist for more than a day in effusions present in the middle ear cleft, even after inoculation with more than 108 genomic equivalents. In contrast, DNA from bacteria that were injected in a viable state is detectable by PCR for weeks. These data thus supported previous studies suggesting that PCR-positive but culture-negative effusions did indeed contain viable bacteria that were not readily cultured (or more likely, were not available for retrieval by lavage). Further, a subsequent report by this same group showing their ability to detect bacterial messenger RNA (which has a half-life measured in seconds to minutes) in culture-negative effusions by reverse transcriptase-PCR (RT-PCR) provided compelling evidence of the existence of metabolically active bacteria in these "sterile" effusions (170).
So the questions remaining were: Where are these viable microbes? Why are they not readily cultured from MEFs? In response, Garth Ehrlich, Chris Post, Bill Costerton, and their colleagues have recently put forth the hypothesis that OM is likely a true "biofilm disease" (49, 165, 166). Biofilms are by nature polymicrobial, they are inherently highly resistant to the action of antibodies and antimicrobials, and they are commonly associated with infections of a persistent nature (Color Plate 3 [see color insert]) (48, 49, 134, 169). This description fits well with that of chronic OM. Post et al. (166) have now demonstrated that a minimally passaged clinical isolate of NTHI can indeed make the transition between planktonic and sessile growth in the environment of the middle ear following transbullar inoculation of a chinchilla. This NTHI strain produced a biofilm on the mucosa lining of the middle ear as observed by scanning electron and confocal scanning laser microscopy (Color Plate 4 [see color insert]). This group continues to investigate the nature, mechanisms, and implications of biofilm production in the middle ear as a contributing factor to both the disease course as well as to the sequelae of OM.
The mechanisms by which biofilm communities resist antimicrobials and antibodies are replete (134); however, one mechanism that is likely to play a role in biofilm persistence in the middle ear is the ability of one microbe to confer antibiotic resistance to other members of the biofilm community because of their close physical association with one another. This phenomenon has been better studied for many of the classic quorum-sensing bacteria to date; however, Budhani et al. (28) showed that, when grown in a continuous culture biofilm system, β-lactamase production by M. catarrhalis protected S. pneumoniae from the action of exogenous β-lactam antibiotics. These microbes are clearly associated with infections of the middle ear and thus the data suggest how synergistic behavior in a mixed microbial biofilm such as this between M. catarrhalis and S. pneumoniae might lead to treatment failure in OM.
Methods of Treatment
We are continually adapting our treatment regimens for OM on the basis of newly acquired information and our improved understanding of the molecular mechanisms behind the pathogenesis of middle ear infections. Some of the ways this increased understanding has led to changes in our approaches to treat and/or prevent OM are reviewed below.
Use of Antimicrobials and Changing Treatment Paradigms
The prescribing of broad-spectrum antibiotics for OM, while still widely used as a treatment regimen, is no longer recommended for use prophylactically in otitis-prone children (73) because of the alarmingly rapid evolution of multiple-antibiotic-resistant bacteria in all three genera of bacteria responsible for OM (25, 56, 64, 65, 74, 84, 160, 172). Nevertheless, antibiotic use in children younger than age 15 for OM remains at a level that is more than 3 times that in any other age group (210). Moreover, approximately 40% of all antibiotic use in children younger than 5 years of age is for treatment of OM (34).
In addition to leading to the emergence of antibiotic resistant organisms, our treatment methods may actually be exacerbating the clinical course of OM. In a recent study by Dagan and colleagues (51), of 19 culture-positive patients in which an organism susceptible to the drug prescribed for treatment was isolated from the initial MEF (H. influenzae, S. pneumoniae, or both) but who also had a resistant S. pneumoniae strain in the NP, they showed that within a few days of treatment, the resistant NP organism had replaced the susceptible middle ear isolate in 47% of these patients. This phenomenon may constitute yet another important mechanism for bacterial superinfection of an ongoing case of OM that is already of mixed microbial etiology. Antibiotic treatment may also exacerbate inflammation in the middle ear. Kawana et al. (116) reported that penicillin treatment seems to augment inflammation in the middle ear during experimental pneumococcal OM while effectively killing S. pneumoniae. Thus, decreasing antibiotic-induced bacterial lysis and the resulting accumulation of cell debris that leads to an increased inflammatory response, an approach that has been used in the treatment of bacterial meningitis, should perhaps be an additional goal for revised treatment paradigms for OM.
As a result of both this rapid emergence of multiple-antibiotic-resistant microbes and many reports that question the efficacy of any antibiotic treatment for chronic OM (50, 72, 214), there has been a call for a change in the overall treatment paradigm for OM (35, 57, 124, 159). Curtailed and more judicious use of antimicrobials for AOM (57), shorter courses of treatment when antibiotic use is warranted (47, 83, 124, 206), deferring antibiotic use in cases of OME or COM, and reserving the use of antibiotics prophylactically for only well-documented cases of recurrent AOM are all measures that have been advocated (35, 159).
Recent studies of 4,860 children in The Netherlands have shown that the vast majority of cases of AOM (>90%) can be managed with only nose drops and analgesics for the first 3 to 4 days. Thereafter, the minority of patients who do not show satisfactory recovery (4 to 5%) can be treated with antimicrobials. In fact guidelines issued by the Dutch College of General Practitioners for the treatment of AOM have recommended since 1990 that for patients 2 years of age or older, treatment should be for symptoms only for the first 3 days (83, 206). Thereafter, patients are reevaluated if symptoms continue and can be treated with a 7-day course of either amoxicillin or erythromycin when indicated. In children 6 months to 2 years of age, the treatment guidelines are the same, except that contact by phone or office visit is mandatory after 24 h. Any child in this latter age group who is acutely ill, or has not improved after 24 h with antimicrobials, is to be referred to an ear, nose, and throat specialist. In the above-cited study of 4,860 children, only two cases of antibiotic-responsive mastoiditis developed and there were no cases of bacterial meningitis; thus, concerns about the risk of serious complications occurring as a result of not treating AOM with antimicrobials appeared to be unfounded. While guidelines such as these may never be adopted globally, the Dutch experience is having an influence on how we think about antimicrobial use for OM. Further changes in treatment paradigms for OME and COM that include the use of antimicrobials may come about as a result of recent discussions about OM as a polybacterial biofilm disease.
Surgical Management of OM
Our increased understanding of the mixed microbial nature of OM as well as information suggesting biofilms involvement in COM also has implications for the surgical management of middle ear infections. For children with chronic or recurrent OM, tympanostomy tubes are often inserted through the tympanic membrane to reduce the symptoms of OM. These tubes do not prevent OM but rather relieve the pressure in the middle ear (and thus most of the painful symptoms of OM) created by the presence of pus and fluid in the middle ear cleft by draining these materials into the auditory canal. Tympanostomy tubes, however, may actually foster the chronicity of OM by providing an inert surface on which an antibiotic-resistant biofilm can form. Biofilms present in the lumen of tympanostomy tubes could serve as a constant source of bacterial cells that can invade the middle ear as well as provide a stimulus for the induction of inflammatory mediators in that site. Saidi et al. (178) recently used a guinea pig model of OM to assay the relative resistance of various silicone- or plastic-based tympanostomy tubes to the formation of a bacterial biofilm. All tympanostomy tubes tested, with the exception of an ion-bombarded silicone tube, showed an accumulated adherent bacterial biofilm by scanning electron microscopy. Thereby, how we design new, or select among available, indwelling devices such as tympanostomy tubes will likely be affected by the contribution of biofilms to the pathogenesis and sequelae of OM.
Treatment of Inflammation Associated with OM
Treatment of the inflammation associated with OM, by targeting the inflammatory mediators involved, has also been discussed as an alternative approach to medical management of OM. However, to date, little efficacy has been shown for decongestants, antihistamines, or specific prostaglandin inhibitors in this regard (30, 70, 112, 113, 135, 191). The use of steroids to ameliorate the inflammatory process in the middle ear has also been tried. Oral prednisolone is only modestly effective as an adjuvant therapy for cases of AOM that have associated discharge through tympanostomy tubes (173). In another study of 210 children with URT infection of 48 h duration or less, wherein half were treated with a placebo and half were treated for 7 days with fluticasone, AOM developed in 38.1% of children in the fluticasone group compared with 28.2% in the placebo group. In children with rhinovirus infection, however, AOM developed significantly more often in children treated with fluticasone (45.7%) than in the placebo group (14.7%). Thus, i.n. fluticasone propionate does not prevent AOM during viral URI in children and may actually increase the incidence of AOM during rhinovirus infection (174). Finally, since most of the proinflammatory cytokines elicited in OM serve as very early markers of infection of the middle ear, several investigators believe that intervention strategies that target these cytokines for pharmacological therapy are unlikely to be successful (140).
Use of Antiviral Agents and Viral Vaccines
Certainly one approach with a great likelihood of success, given the plethora of evidence demonstrating that without the URT viruses bacterial OM is unlikely to develop, is the prevention of OM by treating or immunizing against the viruses associated with bacterial middle ear infections. This could be accomplished by delivery of effective antiviral agents (89) or by vaccination (4, 82, 97). In studies conducted to date, whereas oral rimantadine treatment has not been found to reduce the otological manifestations of influenza in adults or children, i.n. zanamivir and oral oseltamivir were found to significantly reduce the middle ear pressure abnormalities associated with experimental influenza virus infection in adults (89, 90). Preliminary results from a study of oseltamivir treatment in children (171) suggest that this neuraminidase inhibitor also reduces the likelihood of influenza A virus-associated AOM. Other antiviral agents such as tremacamra (a recombinant soluble intercellular adhesion molecule, ICAM-1) (200), AG7088 (a 3C protease inhibitor) (199) or oral pleconaril (a capsid-binding agent) (199) have not been studied in children as effective therapies against OM; however, due to their efficacy in vitro and/or in clinical trials in adults, these studies appear warranted (89).
Some of the strongest evidence in support of both this approach and the critical role of the primary viral predisposing agent in bacterial OM has come as a result of data generated in clinical trials of influenza A virus vaccines. Three trials conducted to date have clearly shown that immunizing children against influenza A decreases the incidence of associated OM. A study conducted in Finland showed that of 187 vaccines, 1 to 3 years of age, and an identical number of matched controls, the incidence of AOM associated with influenza A virus was reduced by 83% in vaccinees (96). The total number of children in the vaccine group with AOM was 35 compared with 55 children in the control group, thus showing a 36% reduction among the vaccinees. Similarly, in a study conducted in eight day care centers in North Carolina with 186 children aged 6 to 30 months, Clements et al. (45) reported that episodes of both AOM and secretory OM were reduced by 32% and 28% in vaccinees, respectively (Tables 3 and 4). In a third study involving 288 children aged 15 to 71 months, receipt of a live, attenuated, cold-adapted, trivalent influenza A virus vaccine i.n. resulted in a vaccine efficacy of 93% against culture confirmed influenza (20). In this study, the vaccinated children also had 30% fewer episodes of febrile OM than the controls.
Despite these results and their predicted cost-effectiveness (132), widespread use of available influenza A vaccines in healthy children has not occurred. Nevertheless, many hope that the anticipated increased acceptability of the i.n. delivered influenza A vaccine will lead to broader use in the targeted pediatric population pending the resolution of a few remaining concerns regarding the safety of delivery of a live vaccine such as this to infants and toddlers. Finally, passive transfer of human immune globulin, enriched with RSV-neutralizing antibodies (RSVIG), has also been shown to confer protection against AOM (186). Children that received high doses of RSVIG developed significantly fewer episodes of AOM than controls. Thus, immunization against the URT viral pathogens that predispose to bacterial OM promises to have a significant impact on the incidence of OM. However, developing effective vaccines for all of the URT viruses predominantly associated with bacterial OM and doing so for a pediatric population (185, 205) are not without many remaining challenges (4, 77).
Use of Vaccines That Target the Bacterial Pathogens of OM
Another approach that is being actively developed in many laboratories is an attempt to immunize (either parenterally or via a mucosal route) against the bacterial pathogens of OM (53, 71, 77, 115, 125, 127, 141, 164). Although many investigators favor the development of mucosal delivery routes for vaccines that target diseases of the upper airway (148), including OM (114, 116, 120), this approach has not yet been fully developed for pediatric diseases to date. This is largely due to some remaining obstacles such as the substantial dilution phenomenon that occurs in the gastrointestinal tract for orally delivered immunogens; the need for mucosally delivered vaccines to be in a particulate form to foster uptake by M cells; the need to protect orally delivered immunogens to ensure their transit through the gut; and when involving replicating agents, overcoming the perceived potential inherent risks, among others (203). Nevertheless, many investigators are working on methods to bypass each of these obstacles for development of mucosally delivered vaccines; this approach may ultimately prove to be the most efficacious for prevention of OM.
In the meantime, however, vaccine candidates designed for a more traditional parenteral delivery route have been or are being developed. These too have a tremendous likelihood of success given that the track record to date for using this approach to prevent other infectious diseases of the respiratory tract has been extremely good. A review of the efficacy of many licensed vaccines currently in use and their ability to act, often via the induction of "herd immunity," is beyond the scope of this chapter; however, Underdown and Plotkin (203) provide an excellent review of the topic. With the goal of preventing OM, the parenteral immunization approach could actually utilize the polymicrobial etiology of OM to its advantage, particularly with regard to the predisposing viral infection-induced inflammation. Serum antibodies transude onto the surface of mucous membranes (15, 68, 158), including into the microenvironment of the middle ear (15, 68) and the NP (203). This transudation of serum components is particularly notable during inflammation. Since viral URT infections typically precede ascending bacterial infection of the middle ear, we can take advantage of the associated inflammation to promote the transudation of specific and protective antibodies (induced by immunization) onto the mucosal surface of the NP, thereby helping to eradicate or reduce the colonizing bacterial load. Antibodies that transude into the middle ear cleft could also be potentially instrumental in eradicating the typically low infecting dose of bacteria that might initially gain access to the middle ear before multiplying to the much higher concentrations that exist in active disease.
This approach has shown tremendous promise in chinchilla models (16, 117) wherein the incidence and severity of OM induced upon challenge with any of several isolates of NTHI could be significantly reduced or eliminated completely by immunization against colonization of the NP using vaccine candidates designed after one of several known NTHI adhesins (Fig. 13). We found that early eradication of NTHI from the NP or even reduction of the bacterial load in that anatomic site by several logs was associated with significant protection against development of OM. Whether or not similar protective efficacy will be induced in children with use of a parenteral approach to target NTHI-induced OM remains to be shown. In this regard, the recent approval and recommendation for use of a polyvalent pneumococcal capsular conjugate vaccine is encouraging. This parenterally delivered heptavalent conjugate vaccine was approximately 97% effective against invasive disease but was also associated with a 6 to 7% reduction in number of episodes of AOM in vaccine recipients compared with controls (22, 62). In a follow up study, efficacy against OM caused by vaccine serotypes of S. pneumoniae was 57% (22, 62).
Summary and Conclusion
It is now widely accepted that many of the URT viruses play a pivotal role in predisposing to infections of the middle ear by either one or more of the three primary bacterial pathogens of OM. The mechanisms by which the viruses mediate their synergistic effect are replete; however, the common theme is compromise or dysregulation of protective functions of the host airway. Many of the methods we have developed to date to medically and/or surgically manage acute and chronic infections of the middle ear have inadvertently contributed to the persistence and recurrence of these infections due to both our lack of complete understanding of the disease process and the incredible adaptability of the microorganisms involved. However, many of these methods have heretofore not necessarily taken into account the polymicrobial nature of the disease, an understanding that is dramatically changing the way that we are currently approaching novel treatment and/or prevention methodologies for OM.
Good clinical evidence exists to believe that by eradicating the predisposing viral infection, one could have a significant effect on prevention of bacterial OM. This goal will not be accomplished easily because of the number of potential viral agents involved, in addition to many other inherent difficulties associated with developing effective viral vaccines; however, progress is clearly being made. Direct blockade of viral attachment and/or uptake via the use of specific antiviral agents is another goal, as is the approach of developing vaccines that target the bacterial commensals residing in the pediatric NP and thus essentially attempting to immunize against colonization. The recent suggestion that the microbes responsible for OM might be inducing the formation of biofilms that are likely to be polybacterial in nature further emphasizes the need to develop novel treatment regimens and vaccines to prevent OM. Vaccine or treatment strategies that effectively inhibit the initial establishment of infection in the middle ear cleft will be particularly important in this regard.
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Lauren O. Bakaletz.
ASM Press, Washington (DC)
Bakaletz LO. Otitis Media. In: Brogden KA, Guthmiller JM, editors. Polymicrobial Diseases. Washington (DC): ASM Press; 2002. Chapter 14.