Clinical assessment of a generic DNA amplification assay for the identification of respiratory adenovirus infections

Mitchell, S.J.; O’Neill, Hugh J.; Ong, Grace; Christie, Sharon; Duprex, W. Paul; Wyatt, D.E.; McCaughey, Conall; Armstrong, V.J.; Feeney, Susan; Metwally, Lobna; Coyle, Peter

doi:10.1016/s1386-6532(02)00082-3

Cited by 21 publications

(20 citation statements)

References 24 publications

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“…Thus, an assay that is able to detect as well as differentiate between AdV species groups such as the Adenoplex can have important clinical relevance in diagnosis and for epidemiological purposes. PCR methods of AdV detection offer a more sensitive and rapid alternative for diagnosis than traditional culture or immunofluorescence (1,18,20). Our data demonstrated that the Adenoplex assay was more sensitive (100%) and had equivalent specificity (100%) compared to viral culture.…”

Section: 69mentioning

confidence: 72%

“…Nested PCR assays have been shown to detect AdV with good sensitivity (1, 18) but involve a two-step PCR procedure increasing the time to complete the assay and chance of carryover contamination. The Adenoplex's sensitivity (at 100 and Ͻ1,000 copies of DNA/ml) is comparable to or better than that of the nested PCR assays, which detected between 400 and 2,500 copies/ml (1) and 640 copies/ml (18), respectively. In addition, the Adenoplex is a single-step PCR and can be completed in less time than a two-step nested PCR assay.…”

Section: 69mentioning

confidence: 85%

“…Recently, PCR-based assays have been employed in clinical practice for the detection of AdV and have proven to be comparable or better than the classic methods (1,4,5,8,11,13,17,18,23). However, many of these assays do not allow for AdV species typing in a rapid and efficient manner.…”

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confidence: 99%

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Rapid Detection and Identification of Human Adenovirus Species by Adenoplex, a Multiplex PCR-Enzyme Hybridization Assay

Pehler-Harrington¹,

Khanna²,

Waters³

et al. 2004

J Clin Microbiol

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Human adenoviruses (AdV) have been implicated in a wide variety of diseases and are ubiquitous in populations worldwide. These agents are of concern particularly in immunocompromised patients, children, and military recruits, resulting in severe disease or death. Clinical diagnosis of AdV is usually achieved through routine viral cell culture, which can take weeks for results. Immunofluorescence and enzyme-linked immunosorbent assay-based techniques are more timely but lack sensitivity. The ability to distinguish between the six different AdV species (A to F) is diagnostically relevant, as infections with specific AdV species are often associated with unique clinical outcomes and epidemiological features. Therefore, we developed a multiplex PCR-enzyme hybridization assay, the Adenoplex, using primers to the fiber gene that can simultaneously detect all six AdV species A through F in a single test. The limit of detection (LOD) based on the viral 50% tissue culture infective dose/ml for AdV A, B, C, D, E, and F was 10 ؊2 , 10 ؊1 , 10 ؊1 , 10 ؊2 , 10 ؊1 , and 10 ؊2 , respectively. Similarly, the LOD for the six DNA controls ranged from 10 2 to 10 3 copies/ml. Twelve common respiratory pathogens were tested with the Adenoplex, and no cross-reactivity was observed. We also validated our assay using clinical specimens spiked with different concentrations of AdV strains of each species type and tested by multiplex PCR and culture. The results demonstrated an overall sensitivity and specificity of Adenoplex of 100%. This assay can be completed in as few as 5 h and provides a rapid, specific, and sensitive method to detect and subtype AdV species A through F.Human adenoviruses (AdV) cause a variety of diseases and are prevalent throughout the world. Common clinical manifestations resulting from AdV infection include pneumonia, cystitis, conjunctivitis, diarrhea, hepatitis, myocarditis, and encephalitis (10). In the general population, AdV often cause mild or self-limiting disease; however, severe disseminated disease can occur in immunocompromised individuals and can be fatal. Pediatric bone marrow transplant patients are at increased risk for AdV infection and have high mortality rates (22). Discontinuation of vaccinating U.S. military trainees against AdV has resulted in a resurgence of respiratory disease epidemics in this population, leading to increased AdV morbidity (12, 15).There are currently 51 recognized AdV serotypes that have been grouped into six different species (formerly subgenera), A to F, based on their physiochemical, biological, and genetic properties (3, 21). Identification of AdV species types can be particularly useful to clinicians, as specific species can cause infections with unique clinical outcomes and epidemiological features. For example, the ability to distinguish between group D AdV, which cause severe and highly contagious keratoconjunctivitis, and group B and E AdV, which cause mild ocular infections, may be of great clinical importance. Also, species typing may help identify disease fro...

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Section: 69mentioning

confidence: 72%

Section: 69mentioning

confidence: 85%

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Rapid Detection and Identification of Human Adenovirus Species by Adenoplex, a Multiplex PCR-Enzyme Hybridization Assay

Pehler-Harrington¹,

Khanna²,

Waters³

et al. 2004

J Clin Microbiol

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“…PCR assays facilitating amplification across variable regions of the hexon gene therefore provide a possible approach to molecular typing. Some of the existing type-specific PCR assays, however, only permit the detection of selected serotypes (11,14,20,27,29,31); others are based on relatively complicated algorithms, including PCR amplification and subsequent digestion with different restriction enzymes (1). The lack of sequence information on all AdV serotypes and the existence of substrains within individual serotypes (6) have prevented the establishment of reliable and economic serotype-specific molecular assays.…”

mentioning

confidence: 99%

Typing of Human Adenoviruses in Specimens from Immunosuppressed Patients by PCR-Fragment Length Analysis and Real-Time Quantitative PCR

Ebner¹,

Rauch²,

Preuner³

et al. 2006

J Clin Microbiol

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“…3). For the timely molecular analysis-based detection of adenoviruses from clinical samples, this methodology compares favorably to generic real-time TaqMan PCR assays (ϳ60 min, excluding DNA extraction) (14), generic direct PCR assays (6 h) (7), and nested PCR assays (6 to 24 h) (25), which have recently been described in published reports. Although each documented method could effectively detect adenoviruses in clinical samples, none presented the ability to determine the adenovirus serotype without further experimentation.…”

Section: Discussionmentioning

confidence: 99%

Use of Oligonucleotide Microarrays for Rapid Detection and Serotyping of Acute Respiratory Disease-Associated Adenoviruses

et al. 2004

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The cessation of the adenovirus vaccination program for military trainees has resulted in several recent acute respiratory disease (ARD) outbreaks. In the absence of vaccination, rapid detection methods are necessary for the timely implementation of measures to prevent adenovirus transmission within military training facilities. To this end, we have combined a fluorogenic real-time multiplex PCR assay with four sets of degenerate PCR primers that target the E1A, fiber, and hexon genes with a long oligonucleotide microarray capable of identifying the most common adenovirus serotypes associated with adult respiratory tract infections (serotypes 3, 4, 7, 16, and 21) and a representative member of adenovirus subgroup C (serotype 6) that is a common cause of childhood ARD and that often persists into adulthood. Analyses with prototype strains demonstrated unique hybridization patterns for representative members of adenovirus subgroups B 1 , B 2 , C, and E, thus allowing serotype determination. Microarray-based sensitivity assessments revealed lower detection limits (between 1 and 100 genomic copies) for adenovirus serotype 4 (Ad4) and Ad7 cell culture lysates, clinical nasal washes, and throat swabs and purified DNA from clinical samples. When adenovirus was detected from coded clinical samples, the results obtained by this approach demonstrated an excellent concordance with those obtained by the more established method of adenovirus identification as well as by cell culture with fluorescent-antibody staining. Finally, the utility of this method was further supported by its ability to detect adenoviral coinfections, contamination, and, potentially, recombination events. Taken together, the results demonstrate the usefulness of the simple and rapid diagnostic method developed for the unequivocal identification of ARD-associated adenoviral serotypes from laboratory or clinical samples that can be completed in 1.5 to 4.0 h.In addition to its effect upon the general population, adenovirus outbreaks within the confined realms of military training facilities pose a special problem that can significantly impair the health and readiness of military personnel. The cessation of a vaccination campaign that successfully prevented outbreaks of adenovirus-associated acute respiratory disease (ARD) in military facilities has resulted in the reemergence of adenovirus-associated ARD epidemics (12,22,30). Furthermore, there are no effective therapeutic or alternate prophylactic treatments for the ARD caused by adenoviruses. To compound the problem, the crowded and stressed situations in military training facilities provide an ideal environment for the airborne transmission of adenoviruses. As a result, the rapid detection of adenoviruses is needed to aid in controlling viral transmission and adenovirus-associated respiratory disease (13, 28).The human adenoviruses are a family of viruses consisting of 51 serotypes (5, 32). Distinction of adenovirus serotypes is based on the neutralization of infectivity, which is type specific and whi...

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Clinical assessment of a generic DNA amplification assay for the identification of respiratory adenovirus infections

Cited by 21 publications

References 24 publications

Rapid Detection and Identification of Human Adenovirus Species by Adenoplex, a Multiplex PCR-Enzyme Hybridization Assay

Rapid Detection and Identification of Human Adenovirus Species by Adenoplex, a Multiplex PCR-Enzyme Hybridization Assay

Typing of Human Adenoviruses in Specimens from Immunosuppressed Patients by PCR-Fragment Length Analysis and Real-Time Quantitative PCR

Use of Oligonucleotide Microarrays for Rapid Detection and Serotyping of Acute Respiratory Disease-Associated Adenoviruses

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