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Journal of Clinical Microbiology, December 2004, p. 5489-5492, Vol. 42, No. 12
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.12.5489-5492.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Evaluation of a Bedside Immunochromatographic Test for Detection of Adenovirus in Respiratory Samples, by Comparison to Virus Isolation, PCR, and Real-Time PCR

Tsuguto Fujimoto,^1* Teruo Okafuji,² Takao Okafuji,² Masahiro Ito,³ Soichi Nukuzuma,³ Masatsugu Chikahira,¹ and Osamu Nishio⁴

Infectious Disease Research Division, Hyogo Prefectural Institute of Public Health and Environmental Sciences,¹ Kobe Institute of Health, Kobe,³ Okafuji Pediatric Clinic, Himeji,² National Institute of Infectious Diseases, Musashi-murayama, Tokyo, Japan⁴

Received 27 May 2004/ Returned for modification 9 July 2004/ Accepted 24 August 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
An immunochromatography (IC) kit for human adenovirus (HAdV) was evaluated with 138 patient nasopharyngeal samples. The samples were collected at a sentinel clinic in Japan from January through June 2003. Patients were diagnosed by clinical manifestation of pharyngoconjunctival fever (n = 38) or exudative tonsillitis (n = 100). The IC kit was positive for 84% (116 of 138) of patients diagnosed at bedside. The remaining extract solution of the IC kit test was transferred into maintenance medium and tested via laboratory diagnoses. The IC kit had 95% sensitivity (116 of 122 patients) with HAdV isolation (isolation) as the standard and 91% sensitivity (116 of 128 patients) with PCR as the standard. All of the IC kit-positive samples were isolation and PCR positive. Similarly, all the isolation-positive samples were PCR positive. Twenty-two IC kit-negative samples were evaluated by real-time PCR. Six samples were IC kit negative and isolation positive and contained 3.8 x 10⁷ to 2.5 x 10⁹ copies of the HAdV genome/ml. Five samples that were only PCR positive contained 3.0 x 10⁴ to 3.8 x 10⁵ copies of the HAdV genome/ml, but one sample was real-time PCR negative. We conclude that the IC kit is a useful bedside diagnostic tool for HAdV infections because it has 95% sensitivity (compared to isolation), but a negative result does not always rule out HAdV infection.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Human adenoviruses (HAdVs) are one of the most commonly isolated viruses and are a significant cause of diseases of the respiratory tract and eye. Pharyngoconjunctival fever (PCF) is a widely known adenoviral disease. HAdVs, particularly types 7 and 3, are also known to cause pneumonia (12, 13). Furthermore, HAdVs are increasingly being recognized as fatal pathogens in immunocompromised patients (1, 2, 5, 18).

Recently, four different kinds of diagnostic methods for HAdV infection became available. The virus isolation technique (isolation) is usually considered the "gold standard" but is time-consuming. PCR is a rapid and sensitive diagnostic technique that can be completed in a few hours, and sequencing the amplified products yields useful data. Real-time PCR for HAdVs has recently become available. Real-time PCR produces results quickly, and quantitative data can be obtained (5, 8, 18). In addition to these laboratory diagnostic techniques, immunochromatography (IC) kits have become available for HAdV diagnosis at the patient's bedside (6, 7, 16, 20, 21).

IC kits for HAdVs have been evaluated in comparison with an isolation technique (6, 20), an enzyme-linked immunosorbent assay kit (16), and PCR (7, 21). However, they have not been evaluated in comparison to the combination of isolation, PCR, and real-time PCR. Quantitative evaluation alone was insufficient; therefore, we evaluated an IC kit in comparison to these three methods. The sensitivity and specificity of the IC kit change according to which method is considered to be the gold standard (15).

The purpose of this study was to evaluate the IC kit, qualitatively and quantitatively, in comparison to multiple, sensitive laboratory diagnostic methods. The reliability and limitations of the IC kit were evaluated. The detection limit of the IC kit was evaluated with HAdV type 1, 2, 3, 5, 6, and 7 isolates.

We found that the IC kit had a higher sensitivity than reported previously, although it was less sensitive than isolation, which was less sensitive than PCR and real-time PCR. However, the IC kit was found to have 95% sensitivity compared to HAdV isolation, the diagnostic technique usually considered the gold standard.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Patients and clinical samples. A total of 138 nasopharyngeal swabs were collected from patients suffering from pharyngoconjunctival fever (n = 38) or exudative tonsillitis (n = 100) during the period of January through June 2003. The mean age of the patients, who ranged in age from <1 to 10 years, was 3.6 ± 0.19 years (mean ± standard deviation [SD]), except for one adult patient who was 38 years old. There was no significant difference in the sex of the patients (72 males and 66 females). All of the patients had temperatures in the range of 38.0 to 40.7°C; the mean temperature was 39.4 ± 0.05°C (mean ± SD). The clinical picture of patients with typical HAdV-associated exudative tonsillitis includes a high fever (>38°C) which persists for about 5 days, and C-reactive protein is usually strongly positive. All clinical samples were collected at the Okafuji Pediatric Clinic, Himeji City, Japan. The protocol was reviewed and approved by the local institutional review board, and informed consent was obtained from all patients.

Clinical diagnosis at bedside. The IC kit used in this study was the SAS Adeno Test (SA Scientific, Inc., San Antonio, Tex.), which was sold under the name Check Ad (AZWELL, Osaka, Japan). The tonsils and posterior pharynx of each patient were vigorously rubbed with a cotton swab moistened in sterile physiological saline. The swabs were extracted with 500 µl of mucolytic agent provided by the manufacturer and rubbed in a soft tube. An aliquot of the extract (200 µl) was filtered and dropped into the IC kit device. Both the test tube and filter were provided by the manufacturer. The IC kit indicated an HAdV-positive result when two colored lines appeared in the device. When only one colored line appeared in the control area of the IC kit, the result was HAdV negative (Fig. 1). The remaining extract solution (about 200 µl) was transferred into a test tube containing 2 ml of Dulbecco's modified Eagles's medium (Sigma) and used as a clinical sample for a later laboratory diagnosis. The test tubes were kept at –80°C until use.

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FIG. 1. Detection of adenovirus in a clinical sample by the IC kit. The test is positive if two colored lines appear in the sample (S) and control (C) areas. The test is negative when only one colored line appears in the control area.

Virus isolation and serotyping. Clinical samples were inoculated onto 80% confluent monolayers of HEp-2 cells in duplicate wells of a 24-well plate (Nippon Becton Dickinson, Tokyo, Japan). For cultivation, Dulbecco's modified Eagle's medium (Sigma) supplemented with 2% heat-inactivated fetal calf serum and antibiotics was used as a maintenance medium. The HEp-2 cells were subpassaged three to eight times to allow time for the cytopathic effect (CPE) to develop. When the CPE became evident, the HAdV isolates were serotyped by a neutralization test (NT) by use of antisera purchased from Denkaseiken (Tokyo, Japan). HAdV type 7 (HAdV 7) used in the detection limit test was isolated and identified in 1998 by the same technique. Enteroviruses were isolated and identified in the same way. Isolates which could not be identified by NT using antiserum for HAdV 1 to 7 were identified by a combination of PCR and sequencing techniques.

Viral DNA preparation. HAdV genomes and enterovirus RNAs were prepared directly from the clinical samples with a QIAamp blood kit (QIAGEN) or a High Pure viral nucleic acid kit (Roche Diagnostics). DNAs from HAdV 1, 2, 3, 5, and 7 isolates were extracted with the High Pure kit.

PCR and sequencing. A single-tube multiplex PCR for HAdVs was carried out as reported previously (3). HAdVs were distinguished by the size of the amplified products. HAdVs which amplified to produce 188- and 301-bp DNA fragments were typed as HAdV 3 and non-subgroup B HAdVs, respectively.

Another primer pair for HAdV 3 (and HAdV 7) was designed and used for PCR and sequencing of the hexon region of HAdVs. The sequences of the primers were 5'-AGAATCATGGACTGATACTGATG-3' (sense) and 5'-AGCCTGTCATTGCCAGGCCAGC-3' (antisense). Amplification reactions were conducted in 50 µl of reaction mixture containing a 0.5 µM concentration of each of the primers, a 200 µM concentration of each dideoxynucleotide, 1.25 U of Taq polymerase (TaKaRa Shuzo, Shiga, Japan), 10 mM Tris-HCl (pH 8.3), 50 mM KCl, and 1.5 mM MgCl₂. The reaction was carried out with a cycle of 94°C for 30 s, 56°C for 30 s, and 72°C for 1 min and was continued for 35 cycles. In the first cycle, the denaturation step continued for 5 min at 94°C, and in the last cycle, the extension step continued for 5 min at 72°C. The expected product size was 1,596 bp (from position 591 to 2186 based on the hexon sequence of HAdV3; GenBank accession number X76549).

Two RT-PCR methods for enteroviruses developed by Ishiko et al. (11) and Oberste et al. (14) were used for enterovirus identification. PCR-amplified products were sequenced directly as reported previously (3, 4, 17). PCR and RT-PCR were performed using a Thermal Cycler Dice (TaKaRa Shuzo). The nucleotide sequence was determined with a model 310 genetic analyzer (Applied Biosystems Japan [ABI]).

Real-time PCR. Real-time PCR for a wide range of HAdVs was used in this study. Primers derived from the highly conserved HAdV hexon 3 and 4 genes by Echavarria et al. (1) were used. The sequences of the primers were 5'-GACATGACTTTCGAGGTCGATCCCATGGA-3' (Hex3) and 5'-CCGGCTGAGAAGGGTGTGCGCAGGTA-3' (Hex4). The expected product size was 140 bp (from position 21589 to 21728 based on the complete sequence of HAdV2; GenBank accession number J01917). One microliter of template DNA was added to a final volume of 25 µl containing 1x SYBR green PCR master mix (ABI) and a 160 nM concentration of the primers Hex3 and Hex4. The real-time PCR was carried out with an ABI PRISM 7900 HT sequence detection system for a cycle of 94°C for 1 min, 57°C for 1 min, and 72°C for 1.5 min, which was continued for 40 cycles. During thermal cycling, the emissions from each sample were recorded and SDS (sequence detection system) software processed the raw fluorescence data to produce threshold cycle (C_T) values for each sample. The SDS software then computed a standard curve from the C_T value of the diluted standards and extrapolated absolute quantities for the unknown samples based on their C_T values. The DNA of the prototype HAdV 2 was amplified by the PCR system of Echavarria et al. (1) as described above. The product (140 bp) was cloned into a pCR2.1 vector and used as a standard.

Detection limit of the IC kit. HAdV 1, 2, 3, 5, and 6 isolated in this study and HAdV 7 isolated in 1998 were diluted in phosphate-buffered saline and tested with the IC kit. Diluted solutions showing slight but clear positivity by the IC kit were used to determine the detection limits of the IC kit. The detection limits were evaluated by determining the 50% tissue culture infective dose (TCID₅₀) per milliliter by use of HEp-2 cells (10) and real-time PCR. Additionally, repeated IC kit detection limit tests were performed with a twofold serially diluted HAdV 3 solution to check the reproducibility. At each concentration, 10 IC kits were tested.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Virus isolation and identification. All the IC kit-positive samples were isolation positive. Six IC kit-negative samples were isolation positive and typed HAdV 1 or 3. Enteroviruses were also isolated individually or as a mixture with HAdVs (Table 1). The IC kit was able to detect HAdVs in coinfected samples. Because enteroviruses grow faster than HAdVs in HEp-2 cells, the CPE of coinfected samples was indistinguishable from that of enterovirus-infected samples. However, the supernatant of coinfected samples was IC kit positive. The IC kit was also useful for confirming HAdV isolation when the HAdV CPE was not clear.

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TABLE 1. IC kit and typing results of detected viruses (n = 138)

PCR, RT-PCR, and real-time PCR. All isolation-positive samples, including IC kit-positive samples, were positive by PCR. However, all HAdV PCR-positive and isolation-negative samples were IC kit negative (Table 1). Samples positive by isolation but IC kit negative had higher concentrations of HAdVs than PCR-positive but isolation-negative samples (Table 2). One isolation-positive sample was coinfected with HAdV (2.1 x 10⁵ copies/ml), and only coxsackievirus type B2 (CB 2) was isolated. Two PCR-amplified products were not clear when stained with ethidium bromide. The two samples did not give concordant results with PCR and real-time PCR. A sample containing HAdV (3.3 x 10⁵ copies/ml) was recorded as PCR negative because only a very weak signal was observed after multiplex PCR. Similarly, one real-time-PCR-negative sample was multiplex PCR positive, but the product band was weak.

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TABLE 2. Results of real-time PCR and typing of the IC kit-negative samples (n = 22)

IC kit for HAdV. The detection limit of the IC kit was 10^1.6 to 10^4.0 TCID₅₀/ml and 10^4.6 to 10^5.8 copies of the HAdV genome/ml. The IC kit was not less sensitive in detecting HAdV 3 and 7 than in detecting HAdV 1, 2, 5, and 6 (Table 3). In repeated IC kit tests, HAdV 3 was positive 10 of 10 times at detection limit concentrations, 3 of 10 times at a twofold serial dilution, and 0 of 10 times at a fourfold serial dilution. A high reproducibility for the IC kit was ascertained.

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TABLE 3. Detection limit of IC kit

Five clinical samples that were IC kit and isolation negative but PCR positive contained 3.0 x 10⁴ to 3.8 x 10⁵ copies of the HAdV genome/ml. However, six clinical samples were IC kit negative in spite of the presence of 3.8 x 10⁷ to 2.5 x 10⁹ copies of the HAdV genome/ml. These samples were all isolation positive, and the IC kit had sensitivity to the isolates.

At the bedside, 40 patient samples were recorded as strongly positive, since in each case a positive colored line appeared before the control line appeared. Seventy-three samples were positive within 10 min. Only three samples required 20 min.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The IC kit has proven to be a useful method for bedside diagnosis of HAdV infections. HAdV infection is difficult to diagnose by the presence of symptoms alone. The sensitivity of the IC kit was 95% in comparison to HAdV isolation in HEp-2 cells. This percentage is higher than that reported earlier by Tsutsumi et al. (20) (72.6%; 69/95). As stated in the manufacturer's guide for the IC kit, the test is highly dependent on the technique of sample collection. We took a great deal of care in sample collection, transportation, and preservation. Additionally, we had more than 10 years of experience in sampling HAdV for isolation before the IC kit became available, which may explain the high detection level. Hara (7) reported a sensitivity of 94.2% (97 of 103 samples) for the same kit in comparison to isolation and that the high sensitivity is dependent on the method of sample collection.

All IC kit-positive samples were isolation positive. Two enterovirus-infected samples were negative by the IC kit and other HAdV diagnoses; the specificity of the IC kit was 100%, as reported previously (6, 20).

In this study, we were able to identify many HAdV-infected patients within a short period of time because a large outbreak of HAdV occurred in Japan in 2003 (13). This outbreak was the largest of its kind in the previous 10 years. The main causative agent was HAdV 3, which accounted for 69% (84 of 122) of the patients diagnosed by NT. According to previous reports, the IC kit is faster than the enzyme-linked immunosorbent assay kit (which takes at least 40 min) and is more useful for detection of respiratory HAdVs (21). Therefore, the IC kit is the only practical bedside diagnostic tool for respiratory HAdV infections.

The IC kit was useful for detecting HAdV in samples coinfected with HAdV and enterovirus. There was no clinical difference between coinfected patients and other HAdV-infected patients. Therefore, HAdV seemed to be the main causative agent of infection in these patients. Coinfection of HAdV3 and HAdV5 could be identified by PCR-based sequencing (17) and use of the HAdV 3- and 7-specific primers designed in this study.

We found that when the SYBR green real-time PCR method was used with isolated HAdV strains, the detection limit of the IC kit was 10^4.6 to 10^5.8 virus genome copies/ml for HAdV 1, 2, 3, 5, 6, and 7. However, there were clinical samples from which we isolated HAdV 3 (four samples) and HAdV 1 (two samples) that were negative by the IC kit despite concentrations of 9.0 x 10⁷ to 2.5 x 10⁹ virus genome copies/ml for HAdV 3 and 3.8 x 10⁷ to 4.5 x 10⁸ virus genome copies/ml for HAdV 1. These clinical samples had virus concentrations within the sensitivity limits of the IC kit; the detection limits for HAdV 3 and HAdV 1 were 10^5.8 and 10^4.6, respectively. According to Hierholzer (9), the bulk of newly synthesized HAdV product is not continuously released but remains cell associated. Only the products that are released into the extraction solution contribute to a positive result by the IC kit. This may explain the 5% false-negative rate.

The IC kit used in this study utilizes a monoclonal antibody made with the HAdV 2 hexon protein. Uchio et al. (21) reported that the IC kit had a lower sensitivity to HAdV 3 and 7 than to HAdV 4, when using serotypes 3, 4, 7, 8, 11, and 37, although these researchers used conjunctival swabs as samples. They reported that the minimum amounts of HAdV 3 and HAdV 8 detected by the IC kit were 4 x 10³ and 4 x 10⁴ PFU, respectively. Shimizu et al. (16) reported that the detection limit of an IC kit was 10^4.45 (2.8 x 10⁴) TCID₅₀/ml for HAdV 3 and that the sensitivities of other IC kits were similar. In our results, the IC kit was sensitive to 10^1.9 to 10^4.0 TCID₅₀/ml for HAdV 1, 2, 3, 5, and 7. Detection limits for the six serotypes were 2.9 ± 1.0 log TCID₅₀/ml and 5.4 ± 0.4 log copies of HAdV genome/ml (mean ± SD). The coefficients of variation were 0.34 and 0.08, respectively. Based on the real-time PCR results, the detection limit of the IC kit was approximately 10^5.4 copies of HAdV genome/ml. In repeated IC kit tests, the high reproducibility of the IC kit was confirmed.

In this study period, no clinical samples containing HAdV 7 were obtained. HAdV 7 is known to cause fatal pneumonic infections in children, including nosocomial infections. We confirmed that the detection limits for HAdV 7 were 10^5.6 copies of the HAdV 7 genome/ml and 10^1.6 TCID₅₀ of HAdV 7/ml. The sensitivity of the IC kit was not lower for HAdV 7 than for the other serotypes in this study. According to Uchio et al. (21), the detection rate of the IC kit is 31% (8 of 26 patients) for HAdV 3 and 60% (3 of 5 patients) for HAdV 7 compared to that of PCR. We were able to detect the presence of HAdV 3 in respiratory samples with the IC kit 93% of the time (in 80 of 86 samples) in comparison to PCR.

The five samples that were IC kit and isolation negative but multiplex PCR positive contained 3.0 x 10⁴ to 3.8 x 10⁵ copies of the HAdV genome/ml. As isolation requires viable virus, PCR was more sensitive than isolation. It is known that many HAdV particles are not infectious, perhaps because they have genomic defects or lack fiber or some other protein (19).

In conclusion, the IC kit is a useful method for diagnosing HAdV diseases at the bedside because it has 95% sensitivity relative to that of isolation. To prevent nosocomial infections, rapid diagnosis at the bedside is necessary. Furthermore, the IC kit provides pediatricians with information about the prognosis of the respiratory diseases at the patient's first visit. However, because the IC kit test had a 5% false-negative rate compared to isolation, careful interpretation is required in cases where IC kit-negative results are obtained and HAdV infection is suspected.

 
We thank M. Yamaoka, T. Kawamura, and Y. Yoshimura for providing the opportunity to do this study.

This study was partly supported by a grant-in-aid for scientific research (15590568) from the Japan Society for the Promotion of Science.

 
* Corresponding author. Mailing address: Infectious Disease Research Division, Hyogo Prefectural Institute of Public Health and Environmental Sciences, 2-1-29, Arata-Cho, Hyogo-Ku, Kobe 652-0032, Japan. Phone: 81-78-511-6640, ext. 236. Fax: 81-78-531-7080. E-mail: Tsuguto_Fujimoto{at}pref.hyogo.jp.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Clinical Microbiology, December 2004, p. 5489-5492, Vol. 42, No. 12
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.12.5489-5492.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Echavarria, M. (2008). Adenoviruses in Immunocompromised Hosts. Clin. Microbiol. Rev. 21: 704-715 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fujimoto, T. Articles by Nishio, O. Search for Related Content PubMed PubMed Citation Articles by Fujimoto, T. Articles by Nishio, O.