The Evaluation of Anti-Adenoviral Therapeutic Agents for use in Acute Conjunctivitis

External ocular infections caused by adenoviruses are among the most common eye infections seen worldwide. They lead to highly infectious community epidemics, seasonal outbreaks, lost productivity, significant patient discomfort and in some cases permanent visual compromise from long-term immune-mediated sequelae1. Though several therapeutic agents have been evaluated for acute viral conjunctivitis in both animal models and human trials, none to date have been approved for therapeutic use in humans2,3,4. Both bacterial and viral pathogens cause acute infectious diseases of the ocular surface with similar clinical presentation. Key differences exist in the mechanism, host response and epidemiology of each etiologic agent. Consideration of these differences shapes our approach to treatment and our approach to the evaluation of therapeutic agents in clinical trials.


Introduction
External ocular infections caused by adenoviruses are among the most common eye infections seen worldwide. They lead to highly infectious community epidemics, seasonal outbreaks, lost productivity, significant patient discomfort and in some cases permanent visual compromise from long-term immune-mediated sequelae 1 . Though several therapeutic agents have been evaluated for acute viral conjunctivitis in both animal models and human trials, none to date have been approved for therapeutic use in humans 2,3,4 . Both bacterial and viral pathogens cause acute infectious diseases of the ocular surface with similar clinical presentation. Key differences exist in the mechanism, host response and epidemiology of each etiologic agent. Consideration of these differences shapes our approach to treatment and our approach to the evaluation of therapeutic agents in clinical trials.

Clinical features of bacterial and adenoviral conjunctivitis
All acute conjunctivities share some common clinical features that aid in the design of appropriate clinical evaluations. Most cases involve conjunctival hyperemia with varying chemosis, some component of ocular discharge and a constellation of symptoms that can include foreign body sensation, pain and itching. A recent evidence-based review 5 examined several databases, including the Cochrane Controlled Trials Register, along with standard ophthalmology texts and concluded that signs and symptoms of acute bacterial and acute viral conjunctivitis are essentially identical. Measurement of the resolution of these symptoms is an essential part of the clinical evaluation of agents for use in acute conjunctivitis of any etiology. Analogy can be made with bacterial conjunctivitis for the clinical signs and symptoms of viral infection, and this analogy can guide in the selection of clinical endpoints. For this reason, the use of similar clinical criteria for one of the primary efficacy endpoints in both viral and bacterial clinical trials is suggested.

Differences in bacterial and adenoviral relationship to the healthy ocular surface
Bacterial conjunctivitis is commonly caused by normal ocular surface flora 6 . When the balance between host defense and microbial colonization on the ocular surface is somehow disrupted, the commensal relationship can proceed to frank infection 7 . Key factors that affect this pathogenic conversion appear to be related both to host defense compromise and specific bacterial species present 8 . In contradistinction to bacteria, adenovirus species are not typically found among the normal ocular flora 9,10,11 . A definitive study employing tissue culture in ocular swab samples obtained from the conjuntival fornicies was unable to demonstrate the presence of adenovirus from even a single sample of over 200 collected in asyptomatic patients presenting for routine eye exam 12 . The absence of adenoviral colonization was similarly demonstrated in conjunctival specimens studied by tissue culture obtained from a series of patients with symptoms of non-infectious keratoconjunctivitis sicca 13 . The presence of adenovirus on the ocular surface would seem to indicate active or recent convalescent infection. This is a critical difference between bacterial and adenoviral conjunctivitis and must be considered when selecting efficacy endpoints for the evaluation of therapeutic agents. Anti-bacterials can clearly demonstrate their clinical utility by simply showing the resolution of clinical signs and symptoms in a shorter time period than would be expected with non-intervention. There is no need to show elimination of bacterial colonization as bacterial sterility is not a feature of the healthy ocular surface. Bacterial conjunctivitis is a much rarer cause of community outbreaks and is less likely to be associated with person-toperson transmission. Described below is the very different behavior of viral conjunctivitis and the relationship between transmissibility and viral shedding.

Viral transmission and shedding
The differences in the transmissibility of bacterial and viral conjunctivitities merit careful consideration. Ocular adenoviral infection represents a significant public health problem in the US and worldwide 14,15 Although exact numbers are difficult to determine, estimates from a US survey of outpatient health encounters 4,9 , comparison with epidemiological surveys completed in outside the US 15 and studies of incidence in military recruits 16 suggest that the number of cases of viral conjunctivitis may be as high as 15-20 million per year in the United States. Adenovirus conjunctivitis is a reportable infection in Germany 17 and is classified as a Category IV infectious disease by Japan's National Epidemiological Surveillance of Infectious Diseases (NESID) with mandated collection, analysis and publication of reports on occurrences 18 . Adenoviral transmission between infected and uninfected hosts is particularly efficient in areas of high population density, overcrowding or poor hygiene 19 . Studies on the rate of 20 horizontal transmission to asymptomatic family members and close contacts suggest transmissibility of up to 50% 21,22,23 . Adenovirus is spread through droplets from the respiratory tract, stool, saliva and tears. Through a process known as viral shedding, infectious particles are transferred from the extracellular environment of lytic infected cells through a variety of fomites 24 . Adenoviral particles, presumably shed from infected patients, have been isolated from multi-dose ophthalmic medications and diagnostic solutions 25,26 Recovery of infectious adenovirus has been reported from samples obtained from inanimate hard surfaces and objects for up to 49 days 27,28 . Actively infected persons readily transmit adenoviruses. Viral shedding persists for 12-14 days after onset of clinical signs and symptoms. Transmission can be prevented by personal hygiene measures including frequent handwashing; cleaning of towels, pillowcases and handkerchiefs; and disposal of contaminated facial tissues. Patients with adenoviral conjunctivitis may shed virus to these objects which can in turn infect other hosts Individuals who work with the public, in schools, or in healthcare facilities in particular should consider a temporary leave of absence from work to prevent infection of others, especially those who are already ill 29 . This is common in hospital and clinic settings and can lead to systemic disease with or without conjunctivitis, particularly in immunocompromised patients 30 . The most effective measures for limiting the severity of adenoviral conjunctivitis outbreaks rely on reducing the contamination of objects, workspaces and surfaces by aggressive steps to remove shed virus particles 31,32 . It follows that reducing shedding at the source -the infected ocular surfacewould be a highly effective strategy for epidemic prevention and control. The clear relationship between shedding virus and infectivity necessarily affects our therapeutic options and our therapeutic requirements in acute viral conjunctivitis. The additional burden is placed on the evaluation of anti-adenoviral agents given their devastating potential to cause outbreaks. Proposed therapeutic agents should aim for a reduction in viral shedding in addition to the resolution of clinical symptoms. An evaluation of efficacy that incorporates both reduced viral infectivity and improvement in symptoms is required to fully demonstrate the utility of any proposed anti-adenoviral therapeutic agent. Similar patterns of epidemic spread, droplet transmission and shedding are not typical features of bacterial conjunctivitis, though outbreaks have been reported in humans 33 and vector-dependent spread confirmed in animal models 34,35,36 . Bacterial conjunctivitis is a much less likely cause of outbreaks and is not a significant public health challenge (we acknowledge the enormous importance of bacterial conjunctivitis caused by C. Trachoma and defer its discussion as it is more commonly a chronic, endemic, recurrent infection with a distinct clinical course) 37 . Though vertical transmission remains an important aspect of neonatal bacterial conjunctivitis, these cases are rare in the industrialized world and do not share the features of epidemic infection. Furthermore neonatal conjunctivitis passed intrapartum from mother to newborn is easily eliminated through ocular administration of povidone-iodine at the time of birth 38 .

Ocular immune response in bacterial and adenoviral infections
Components of both the innate and adaptive acquired immune systems play important roles in ocular defense. 39 While the predominantly extracellular bacterial pathogens are more effectively controlled by the innate ocular defense mechanisms, viral infections often lead to a more prolonged course. Viral exposures frequently involve a more robust acquired immune cascade with significant inflammatory damage 4041,42 . It is precisely this exuberant immune reaction that leads to the signs and symptoms of viral conjunctivitis and immune-mediated sequelae. It is often clinically beneficial to temper the ocular immune response in both viral and bacterial infections, with topical steroids frequently the agents of choice. Steroids have well characterized effects on both innate and adaptive immunity. The features of the immune responses to viral and bacterial pathogens need to be considered along with the relative effects of steroid on each system: Steroids have a more dramatic inhibitory effect on the adaptive system, and this is precisely the system that is most important at eliminating viral infections. In expected that steroids would have less of an effect on the eye's ability to counter bacterial pathogens than they would on the elimination of viral organisms. It has been demonstrated that co-administration of potent topical steroids along with antibiotics does not lead to higher bacterial counts (measured as CFU's) 43 in the normal bacterial conjunctival flora.
It has been repeatedly shown in ocular adenoviral infection that use of topical steroids can prolong the duration of viral shedding and therefore lengthen the period of transmissibility in these cases 44 . It is for this reason that topical steroid monotherapy in ocular adenovirus infections is ill-advised. It is well known that a short course of topical corticosteroids (and in some severe cases oral steroids) can limit patient discomfort and prevent some immunerelated inflammatory complications of acute viral conjunctivitis. While this strategy may have some efficacy in the short-term amelioration of symptoms, even a short course of relatively low-potency corticosteroids without the addition of a suitable anti-viral agent can increase the duration of viral shedding and prolong the infectivity of affected patients 45 . The addition of topical steroids cripples the eye's immune response to viral pathogens. The effect on the ability to effectively clear viral infections is so pronounced that the addition of topical steroids can even reverse the effect of the most potent anti-virals 46 . This in turn can potentiate the occurrence of community outbreaks and epidemic transmission in schools, places of business and medical facilities 47 . As described above, this additionally requires that effects on infectivity be considered along with symptom resolution in the clinical evaluation of anti-adenoviral therapies 48 .

Detection of adenoviral infectivity
There are several techniques available for the detection of adenovirus from ocular specimens. Despite recent advances in nucleic acid-based detection and the availability of a rapid point-of-care immunochromatographic tests for the presence of specific viral components, cell culture remains the only reliable method for the demonstration of viable, infectious virus. Cell culture with confirmatory immunofluorescence (CC-IFA) is a highly sensitive and specific test and is considered the "gold standard" for the recovery of infectious virus from ocular samples. CC-IFA requires the presence of infectious virus and demonstrates unequivocally the ability of the recovered virus to cause a cytopathic effect (CPE) in a living cell. When combined with immunoflourescent staining, it provides a means to determine the presence, infectivity and identity of a viral specimen. A sample from a conjunctival swab is inoculated in susceptible cell line and followed over time to measure the cytopathic effects (CPE). The "Shell Vial Culture" method is a specific cell culture technique that enables more rapid identification of CPE 49 . This test utilizes shell vials, centrifugation and visualization of adenovirus proteins inside host cells through binding of fluorescent dye. Shell vials are glass culture tubes that contain a coverslip coated with an A549 cell monolayer. The culture tube is inoculated with the clinical specimen and the tube is centrifuged at low speed and incubated. It is hypothesized that the centrifugation enhances the adenoviral entry into the susceptible cells. The visualization technique is indirect, where a secondary antibody labeled with fluorochrome is used to recognize a primary antibody directed towards a conserved adenoviral epitope. This test significantly shortens the time requirement and enhances the sensitivity and specificity. Positive results can be obtained from the visualization of even a single brightly stained cell, confirming that the adenoviral particles were capable of entering a cell, uncoating, replicating and producing infectious prodigy virions. In this way CC-IFA in general and the Shell Vial method specifically provide an unequivocal way to determine the infectivity of an ocular specimen. It is for this reason that we propose assessment of infectivity by CC-IFA as a second primary endpoint for clinical trials designed to evaluate the efficacy of antiadenoviral therapeutic agents.

Proposed clinial study design for demonstrating utility of therapeutic agents in acute adenoviral conjunctivitis
The ideal treatment for adenoviral conjunctivitis would alleviate patient symptoms, resolve clinical signs, decrease inflammatory damage, shorten the clinical course of infection, reduce the duration of viral shedding and decrease the period of infectivity. The evaluation of all therapeutic agents for use in adenoviral conjunctivitis should include analysis of clinical and infectious parameters and consider effects on the individual patient and the community as a whole. The use of separate primary efficacy endpoints is proposed that can demonstrate the following: 1. Resolution of signs and symptoms associated with viral conjunctivitis. 2. Decrease in infectious viral shedding measured by CC-IFA at the test-of-cure visit. Resolution of signs and symptoms of the disease is the most clinically meaningful assessment and derives from the similar clinical features shared by acute bacterial and acute viral conjunctivitis. Particularly from the patient's individual perspective, the resolution of signs and symptoms is the most important clinical outcome. Much can be learned and www.intechopen.com Conjunctivitis -A Complex and Multifaceted Disorder 132 borrowed from the myriad experience gained over decades of clinical trials in bacterial conjunctivitis. The use of standardized conjunctivial grading, scaled scoring for ocular discharge and conjunctival injection all have application in both bacterial and viral disease. The required analysis of viral shedding, which derives from the differences in transmission between bacterial and viral conjunctivitis, is important to ensure that symptomatic relief in individuals doesn't lead to prolonged infectivity. The ideal therapeutic will radpidly decrease viral loads and shorten the overall length of time that active, replicating virus can be isolated from the ocular surface. This will ensure that the simple masking of symptoms cannot be substituted for a true viral cure. Though indivdual subjects may improve on symptomalleviating therapy only, the requirement to reduce infectivity should ensure that no agents gain approval that could potentially lengthen epidemics or threaten the public health. The requirement for all proposed agents to satisfy both of these endpoints is the most effective way to ensure that proposed ant-adenoviral therapies address both the infectious and inflammatory consequences of the disease.