Evaluation of the shell vial technique for detection of ocular adenovirus

doi:10.1016/S0161-6420(99)00718-6

Ophthalmology

Volume 106, Issue 7, 1 July 1999, Pages 1324-1327

Presented in part as a poster at the Association for Research in Vision and Ophthalmology annual meeting, Fort Lauderdale, Florida, May 1998, and as a paper at the Ocular Microbiology and Immunology Group Meeting, New Orleans, Louisiana, November 1998.

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Abstract

Purpose

The shell vial technique is a cell culture method that uses centrifugation and immunofluorescence to decrease the time required for a positive test. The authors evaluated the shell vial technique as a diagnostic test to detect adenovirus in conjunctival specimens of patients with adenoviral conjunctivitis.

Design

Retrospective and prospective case series.

Participants

Forty-six patients with adenoviral culture-positive ocular infection.

Methods

The minimum time of incubation (days) that was required for testing clinical isolates with the shell vial was determined with adenovirus serotypes 5 and 8. In a masked retrospective study, 25 true-positive (frozen clinical samples) and 25 true-negative specimens were tested for the presence of adenovirus using the shell vial technique. The 25 true-negative samples included herpes simplex virus, Chlamydia trachomatis, Haemophilus influenzae, Streptococcus pneumoniae, and Staphylococcus aureus. In a prospective study, 21 patients who later tested positive in cell culture for adenovirus were concurrently tested with shell vial.

Main outcome measures

The time of incubation was determined in days, and the sensitivity, specificity, positive and negative predictive values, and the efficacy of the shell vial test were determined.

Results

The minimal time of incubation for testing ocular samples by shell vial was 3 days. In the retrospective study, the sensitivity, specificity, positive predictive value, negative predictive value, and efficacy were 92%, 100%, 100%, 93%, and 96%, respectively. Comparably (P = 0.99), in the prospective study the sensitivity, specificity, positive predictive value, negative predictive value, and efficacy were 95%, 100%, 100%, 96%, and 97%, respectively. The shell vial (93%, 43 of 46) was equivalent (P = 0.42) to cell culture (100%, 46 of 46) for detecting adenovirus, but a positive result was obtained in significantly less time (3 days versus 9.41 ± 6.23 days) (P = 0.00001).

Conclusions

The shell vial technique was found to be a definitive method for identifying adenovirus from ocular specimens. A clear benefit for the ophthalmologist is that the test can provide a faster positive result (3 days) compared with conventional cell culture, which can take 1 to 3 weeks for adenovirus isolation.

Section snippets

Shell vial technique

The shell vial technique for adenovirus isolation was performed by a previously established protocol.9 A brief description of the procedure in this study is as follows:

Growth medium was removed from shell vials containing a coverslip layered with A549 cells. The A549 cells were washed with phosphate buffered saline (PBS) containing magnesium and calcium (Bartels Inc., Issaquah, WA). The shell vials were inoculated with 0.2 ml of each respective specimen and centrifuged 1 hour at 1000 g. One ml

Results

The minimum time of incubation that was required for positive staining for all titers and both serotypes was 3 days. Positive staining was present for adenovirus serotype 5 at all titers (pfu/vial) and days except for 10⁰ pfu/vial on day 1. Adenovirus serotype 8 did not stain positive for any titer on day 1 and did not stain positive for titers 10¹ and 10⁰ on day 2.

Table 1 describes the results of the shell vial technique for detection of adenovirus from conjunctival specimens. The 25

Discussion

A positive laboratory test for ocular adenoviral infection is a function of exceeding a threshold level of viral DNA, infectious virions, or viral antigen. The earlier a specimen is obtained during symptomatic ocular adenoviral infection, the larger the amount of viable virus and antigen is present in the conjunctiva. As the time between the onset of clinical symptoms and the time to obtaining a clinical sample increases in days, the amount of available adenovirus decreases. The rapid

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Cited by (19)

Adenoviral keratoconjunctivitis
2015, Survey of Ophthalmology
Citation Excerpt :
Testing includes cell culture, PCR, direct immunofluorescence, and rapid antigen detection immunoassays. Viral cell cultures of the conjunctival specimen allow confirmation of the adenovirus with immunofluorescence, but are less commonly performed because of the necessity for elaborate equipment, trained laboratory personnel, and the significant delay in obtaining results.16,23,54,60,110 Detection of adenoviral DNA in conjunctival specimen by PCR is more commonly used, but is a complex process that also requires expensive equipment and technical expertise.14,15,24,52,53
Viral conjunctivitis caused by adenovirus is the most common infectious conjunctivitis. Adenoviruses are highly contagious pathogens. The modes of transmission are mainly through hand to eye contact, ocular secretions, respiratory droplets, and contact with ophthalmic care providers and their medical instruments. The most frequent manifestation of ocular adenoviral infection is epidemic keratoconjunctivitis, followed by pharyngoconjunctival fever. Epidemic keratoconjunctivitis is also the most severe form and presents with watery discharge, hyperemia, cheosis, and ipsilateral lymphadenopathy. Pharyngoconjunctival fever is characterized by abrupt onset of high fever, pharyngitis, bilateral conjunctivitis, and periauricular lymph node enlargement. Isolated follicular conjunctivitis without corneal or systemic involvement also occurs. The rate of clinical accuracy in diagnosing viral conjunctivitis is less than 50%. Rapid diagnostic tests now being used decrease unnecessary antibiotic use. Treatment for viral conjunctivitis is mostly supportive. The majority of cases are self-limited, and no treatment is necessary in uncomplicated cases.
A comparison of methods for detecting adenovirus type 8 keratoconjunctivitis during a nosocomial outbreak in a Neonatal Intensive Care Unit
2003, Journal of Clinical Virology
Background: An outbreak of epidemic keratoconjunctivitis (EKC) due to adenovirus (Ad) type 8 and involving 14 members of the hospital staff and 33 neonates admitted to the Neonatal Intensive Care Unit of the local University Hospital occurred between September and December 2000 in Pavia, Italy. The outbreak was preceded by an outbreak of EKC within the community. Objective: To compare the performance of conventional virus isolation on cell cultures, direct detection of Ad antigens in conjunctival cells by a direct fluorescent assay (DFA) and Ad DNA detection in conjunctival swabs by polymerase chain reaction (PCR) for diagnosis of adenoviral conjunctivitis. Study design: Of conjunctival swabs collected from 47 patients, all were tested by virus isolation, 43 by direct Ad antigen detection, and 37 by Ad DNA detection. Direct Ad antigen detection was carried out by DFA using a group-specific monoclonal antibody. Detection and subgrouping of Ad DNA by nested PCR was performed using two sets of primers complementary to hexon and fiber genes, respectively. Results: Ad was detected in 24/47 (51.1%), 21/43 (48.8%), and 23/37 (62.1%) samples by virus isolation, direct antigen detection and PCR, respectively. Overall, 30/47 (63.8%) samples were Ad-positive. Of 37 specimens tested in parallel by all three methods, Ad was detected by at least one of the three techniques in 26/37 (70.3%). All Ad isolates were identified as serotype 8 by neutralization, while all PCR-positive samples were identified as belonging to subgroup D. No other virus was isolated from any conjunctival swab. Time required for test completion was 9.6 (4–20) days for virus isolation, 1–2 h for DFA and 24 h for PCR. Conclusions: DFA was a sensitive and rapid assay but results depend on the quality of sample and the expertise of the observer. PCR was the most sensitive assay, although it takes longer to perform and requires dedicated facilities; thus, it could be restricted to DFA-negative samples. Virus isolation is still useful from an epidemiological point of view.
The disinfection of contact lenses contaminated with adenovirus
2001, American Journal of Ophthalmology
PURPOSE: We compared the efficacy of different contact lens disinfection systems to eliminate adenovirus.
METHODS: Laboratory study evaluating the elimination of adenoviral ocular isolates by contact lens disinfection systems. Hard (gas permeable) and soft contact lenses were contaminated with adenovirus serotypes 8 and 19, and then they were disinfected with chemical, hydrogen peroxide, and heat sterilization systems. The survival of the adenovirus was determined by the shell vial technique.
RESULTS: Adenovirus survived chemical and hydrogen peroxide disinfection but not heat sterilization.
CONCLUSION: Because heat sterilization is not readily available to sterilize adenovirus contaminated contact lenses, it may be prudent for patients with adenoviral keratoconjunctivitis to dispose of unclean contact lenses.
The Diagnostic Potential of Monoclonal Antibodies to Adenovirus
2022, Applied Biochemistry and Microbiology
Enhanced detection of viruses in cell cultures
2022, Manual of Molecular and Clinical Laboratory Immunology: 7th edition
Viral disease of the eye
2022, Clinical Virology: Third Edition

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^☆: Supported by The Pennsylvania Lions, Sight Conservation and Eye Research Foundations, Inc., The Eye and Ear Foundation of Pittsburgh, Pittsburgh, Pennsylvania, a core grant for vision research (EY 08098) from the National Eye Institute, Bethesda, Maryland, and Research to Prevent Blindness, Inc., New York, New York.

¹: The authors have no proprietary interest in any of the products presented in this study.

^∗: The community ophthalmologists who provided the patients and medical history information are listed in the Appendix at the end of the article.

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Evaluation of the shell vial technique for detection of ocular adenovirus☆

Abstract

Purpose

Design

Participants

Methods

Main outcome measures

Results

Conclusions

Section snippets

Shell vial technique

Results

Discussion

Ophthalmology

Ophthalmology

Ophthalmology

Adenovirus keratoconjunctivitis

A 5-year evaluation of the Adenoclone test for the rapid diagnosis of adenovirus from conjunctival swabs

Cornea