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Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv. Short communication. Performance of a new immunochromatographic assay for ...

Journal of Clinical Virology 44 (2009) 173–175

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Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv

Short communication

Performance of a new immunochromatographic assay for detection of adenoviruses in children夽 Fatma Levent a , Jewel M. Greer a,b , Malorie Snider a,c , Gail J. Demmler-Harrison a,b,∗ a

Baylor College of Medicine, Department of Pediatrics, Section of Infectious Diseases, Houston, TX, United States Diagnostic Virology Laboratory, Texas Children’s Hospital, Houston, TX, United States c Harvard University, Boston, MA, United States b

a r t i c l e

i n f o

Article history: Received 17 June 2008 Received in revised form 3 November 2008 Accepted 4 November 2008 Keywords: Rapid assay Adenovirus Diagnosis Children

a b s t r a c t Background: Adenoviruses are a prominent cause of respiratory, ocular, gastrointestinal, and disseminated diseases in healthy and immunocompromised children. An accurate rapid diagnostic assay may impact clinical decision-making. Objectives: Evaluate the performance of a new rapid assay for detection of adenoviruses directly in pediatric clinical specimens. Study design: The rapid assay was performed on adenovirus culture-positive original samples and on an equal number of culture-negative samples matched by patient age and specimen type. Discrepant results were resolved using a polymerase chain reaction (PCR) assay. Results: 200 adenovirus culture-positive and 200 adenovirus culture-negative samples were evaluated from 315 different patients. Overall sensitivity was 55% and specificity was 98.9%. The assay was most sensitive in children 5 years old and younger and most specific in respiratory samples. Conclusions: The rapid assay was highly specific for detecting adenovirus infections in children. However, since this rapid assay had only moderate to low sensitivity, samples with negative rapid assay results should have additional testing for adenovirus performed by either viral culture or PCR. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Adenoviruses are a prominent cause of respiratory, ocular, gastrointestinal, and disseminated diseases in healthy and immunocompromised infants, children and adults.1–4 Infection with adenoviruses can mimic Kawasaki disease and bacterial infections, leading to unnecessary treatments if not properly diagnosed.5 Community and health-care associated outbreaks also occur and prompt isolation of infected patients is critical for outbreak containment.3,6,7 Adenovirus may be detected using viral culture, direct fluorescence assay (DFA), or molecular methods, all of which require special expertise or equipment to perform and hours to days to provide final results.8–13 Furthermore, DFA and even the newer respiratory virus panels or multiplex PCR assays may not be sensitive methods for detection of many common serotypes of adenovirus.13

夽 Presented in part at Infectious Diseases Society of America 45th Annual Meeting, San Diego, CA, October 4–7, 2007. ∗ Corresponding author at: Texas Children’s Hospital, Mail Code 3-2371, 6621 Fannin Street, Houston, TX 77030-2399, United States. Tel.: +1 713 436 9572; fax: +1 713 798 7249. E-mail addresses: [email protected] (F. Levent), [email protected] (J.M. Greer), [email protected] (M. Snider), [email protected] (G.J. Demmler-Harrison). 1386-6532/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2008.11.002

A new qualitative in vitro immunochromatographic membranebased assay that utilizes a specific monoclonal antibody against the group-reactive hexon antigen common to all known serotypes of human adenovirus was evaluated. Adenovirus antigen was detected directly in clinical samples, in as little as 15 min,14 timely enough to potentially influence clinical decision-making. 2. Methods Specimens submitted to the Diagnostic Virology Laboratory at Texas Children’s Hospital from January through December 2006 for virology testing were evaluated using the SAS Rapid Adeno test (SAScientific, San Antonio, TX, USA) 200 adenovirus culture positive and 200 adenovirus culture negative samples obtained from 315 pediatric patients were evaluated. The rapid assay was performed per manufacturer’s instructions15 and results were interpreted by the presence or absence of visually detectable colored lines (Fig. 1). Following a brief extraction step using a buffer containing 0.1% sodium azide, the sample was added to a sample well in a plastic device. An immobilized capture antibody on a membrane formed a colored line at the specimen “S line” if the specimen contained adenovirus antigen. An internal control line C, containing antimouse antibody that captured the colored conjugate antibody, was also built into the device to serve as a procedural quality

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Fig. 1. SAS Rapid Adeno test kits showing testing wells, sample (S) and control (C) lines.

control to assure the sample migrated properly and that the test was performed properly. Quality control measures included using internal kit procedural controls, as well as external laboratory positive (adenovirus-positive cell culture suspension in maintenance media) and negative (uninoculated cell culture suspension in maintenance media) controls for each test kit run. The test was considered positive if a visible line was present at both the S and C positions, and considered negative if a visible line was present at the C position and absent at the S position. If no lines were present, the test was considered invalid and repeated. All positive and negative readings were considered final after 15 min of incubation. All specimens were inoculated onto three cell lines [human foreskin fibroblasts, rhesus monkey kidney and human lung carcinoma (A549)], examined daily under light microscopy for evidence of viral CPE. Virus identification was confirmed using a virus specific DFA (D3Ultra DFA Respiratory Virus Screening and ID kit; Diagnostic Hybrids, Inc. Athens, OH). Discrepant results were resolved using a highly sensitive, user-developed, traditional, single sample PCR assay that detected a conserved 161 bp region of the adenovirus serotype 2 hexon gene and detected all known adenovirus serotypes including noncultivatible enteric Ad40/Ad41 serotypes.16 Sensitivity, specificity, and predictive values were calculated using standard 2X2 tables. Chi-square and Fisher’s exact test was used to evaluate the significance of the performance of the rapid assay. A p value of 5 yr Co-infected specimens

151 49 168 32 8

151 49 166 34 9

F. Levent et al. / Journal of Clinical Virology 44 (2009) 173–175

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Table 3 Performance of the SAS Rapid Adeno test compared to viral culture for detection of adenovirus directly in clinical samples in pediatric patients. Rapid assay

% Sensitivity, (CI)a

% Specificity, (CI)a

PVb (CI)a

PVc (CI)a

Overall performance Respiratory Non-respiratory Age ≤5 Age >5 Co-infected

55 (48.1–61.9) 56.2 (48.4–64.2) 53 (39.1–67) 59.5 (52.1–66.9)* 31.2 (15.2–47.3) 25(3–65)

99 (97.6–100) 100(99–100) 95.9 (90.4–100)* 99.4 (96–99.8) 97(85–99) 100(66–100)

98.2 (95.8–100) 100(99–100) 92.9 (83.3–100) 99 (97.1–100) 90.9 (73.9–100) 100 (19.8–100)

68.8 (60.2–77.3) 69 (59.8–79.4) 67.1 (49.7–84.5) 70.8 (61.9–79.7) 60(31–89) 60(4–99)

a b c *

CI, 95% confidence interval. PPV, Positive predictive value. NPV, Negative predictive value. p < 0.05.

4. Discussion The high specificity, but moderate to low sensitivity, of the rapid adenovirus assay suggests negative specimens should have additional testing performed by either culture or molecular methods.13,21,22 Previous studies of the rapid adenovirus assay, performed mostly on respiratory specimens from adults, have shown comparable specificity (91–100%), and variable sensitivity (54.7–95%).14,15,17–19 . A different rapid assay (RPS Adeno Detector Test, Rapid Pathogen Screening, Inc., South Williamsport, PA), licensed for eye specimens only, is reported to have a higher sensitivity (88%), and slightly lower specificity (91%) compared to the SAS Rapid Adeno test.20 The current SAS Rapid Adeno test reported in our study was significantly more sensitive in samples from children 5 years of age or younger, the age range with the highest incidence of adenovirus infection.1 The sensitivity of the rapid test also may vary by specimen type, but the numbers available in our study were not large enough to evaluate statistically. Sensitivity may vary by adenovirus serotypes, and further studies are needed to adequately address this important issue. It also is possible that the initial sample extraction step using a buffer may dilute the viral antigen in some specimens and influence sensitivity. Studies evaluating the performance of the test directly on clinical samples, without use of buffer, or development of a more sensitive assay are therefore indicated. The rare apparent false positive results obtained when the rapid test was compared to culture may have been due to the presence of enteric adenovirus serotypes, such as Ad40 and Ad41, which are not routinely cultivatable in viral cell culture, but may be detected by the rapid test and PCR assay. If so, this rapid test may provide an advantage over cell culture in samples containing these noncultivatible enteric adenoviruses. The SAS Rapid Adeno test was also easy to perform, and provided results in 15 min, a time frame that is useful in clinical decision making, when the test is positive. Further studies evaluating the use of this test outside the virology laboratory, such as in point-of-care settings, may be of interest to clinicians.13,21,22 Acknowledgement This study was supported, in part, by materials donated by Remel, Inc., Lenexa, KS. References 1. Demmler GJ, Adenoviruses. In: Long S, Pickering L, Prober C, editors. Principles and practice of pediatric infectious diseases. 4th ed. Philadelphia, PA: Churchill Livingstone; 2008. p. 1076–80. 2. Munoz FM, Piedra PA, Demmler GJ. Disseminated adenovirus disease in immunocompromised and immunocompetent children. Clin Infect Dis 1998;27:1194–200.

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