J Infect Chemother 20 (2014) 303e306
Contents lists available at ScienceDirect
Journal of Infection and Chemotherapy journal homepage: http://www.elsevier.com/locate/jic
Original article
Evaluation of new immunochromatographic assay kit for adenovirus detection in throat swab: Comparison with culture and real-time PCR results Miyuki Morozumi a, b, Hideaki Shimizu c, Yuki Matsushima c, Keiko Mitamura d, Takeshi Tajima e, Satoshi Iwata b, Kimiko Ubukata, PhD a, b, * a
Laboratory of Molecular Epidemiology for Infectious Agents, Kitasato Institute for Life Sciences, Kitasato University, Tokyo, Japan Department of Infectious Diseases, Keio University School of Medicine, Tokyo, Japan Division of Virology, Kawasaki City Institute for Public Health, Kanagawa, Japan d Department of Pediatrics, Eiju General Hospital, Tokyo, Japan e Department of Pediatrics, Hakujikai Memorial Hospital, Tokyo, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 September 2013 Received in revised form 10 January 2014 Accepted 14 January 2014
A new immunochromatographic (IC) assay kit, BD Veritor System Adeno was evaluated to comparing with commercial available kit, BD Adeno Examan, cell culture, and real-time PCR using throat swab samples. Specimens were collected from 146 pediatric patients between July 2011 and January 2012. Mean age of patients was 4 years (8 monthse15 years old). Patients were diagnosed with pharyngitis (n ¼ 67), tonsillitis (n ¼ 45), pharyngoconjunctival fever (n ¼ 26), upper respiratory tract infection (n ¼ 6), conjunctivitis (n ¼ 1), or bronchitis (n ¼ 1). Thirty-one of the patients (21.2%) had more than one disease. Among all samples, 61 (41.8%) were positive for adenovirus with BD Veritor System Adeno; 68 (46.6%) with BD Adeno Examan; 63 (43.2%) with real-time PCR; and 65 (44.5%) with cell culture. Serotype 3 (n ¼ 41; 63.1%) was predominant among the 65 adenovirus isolates, followed by serotype 2 (n ¼ 12; 18.5%), 1 (n ¼ 6; 9.2%), 5 (n ¼ 4; 6.2%), and 4 (n ¼ 2; 3.1%). Relative sensitivity and specificity of BD Veritor System Adeno, BD Adeno Examan, and real-time PCR were 93.8% and 98.7%, 96.9% and 93.8%, and 96.9% and 100%, respectively. Positive predictive and negative predictive values for these methods were 98.4% and 95.1%, 92.6% and 97.4%, and 100% and 97.6%, respectively. The sensitivity and specificity of real-time PCR was greater than that of IC assay kits. However, IC assay kits also showed high sensitivity and specificity appropriate for clinical use. Ó 2014, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Keywords: Adenovirus Rapid detection Immunochromatographic assay Real-time PCR Serotype
1. Introduction Human adenoviruses (HAdV) are major causes of respiratory tract infections [1e3]. The most common adenoviral infections are pneumonia, bronchitis, pharyngitis, tonsillitis, conjunctivitis, and pharyngoconjunctival fever (PCF). Currently, 7 HAdV species designated have been identified A to G. These species include more than 50 serotypes [4e6]. HAdV serotypes have been characterized according to illness caused: are serotypes 1 to 7 are related to upper * Corresponding author. Department of Infectious Diseases, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Tel.: þ81 3 3353 1211; fax: þ81 3 5363 3711. E-mail address: [email protected] (K. Ubukata).
respiratory tract infection; serotypes 3, 7, 14, and 21, to lower respiratory tract infection; serotypes 3, 4, and 7, to PCF; and serotypes 8, 19, and 37, to epidemic keratoconjunctivitis (EKC) [3,5,7]. Standard diagnostic methods for adenoviral infections include cell culture and antigen detection [8e10]. Although adenovirus can be isolated by cell culture considerable time is required. Commercially available rapid diagnostic kits using immunochromatography (IC) or enzyme-linked immunosorbent assay (ELISA) have been developed for early diagnosis of adenoviral infections [11e14]. Meanwhile, conventional PCR or real-time PCR can detect adenoviral DNA rapidly and also can obtain quantitative results with high sensitivity and specificity [15e19]. Early detection of HAdV is important for both guiding treatment and preventing spread of the infection.
http://dx.doi.org/10.1016/j.jiac.2014.01.005 1341-321X/Ó 2014, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
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M. Morozumi et al. / J Infect Chemother 20 (2014) 303e306
To evaluate of a new rapid diagnostic kit, BD Veritor System Adeno, we compared the results of cell culture, IC assay kits (BD Veritor System Adeno; Becton and BD Adeno Examan; Dickinson and Company, Franklin Lakes, NJ), and real-time PCR using throat swab samples from pediatric patients. 2. Materials and methods 2.1. Patients and clinical samples Eleven medical institutions participated actively in this study from July 2011 to January 2012. All together, 146 clinical samples were collected from pediatric outpatients diagnosed with pharyngitis, tonsillitis, or conjunctivitis likely caused by HAdV based on clinical symptoms and findings such as fever (temperature exceeding 37.0 C), pharyngeal pain, tongue fur, and conjunctivitis. Throat swab samples were collected using universal transport media (Becton, Dickinson and Company) for cell culture. After informed consent was obtained from patients, parents, samples were collected by pediatricians. Mean age of patients was 4 years (8 monthse15 years old); 88 were male. 2.2. Real-time PCR to detect adenovirus Detection of HAdV DNA in specimens was carried out by realtime PCR. Clinical samples were centrifuged at 2000 g at 4 C for 10 min to collect virus attached to cells. One hundred and fifty microliter containing pellet after the supernatant discarded was mixed gently. The supernatant was used for cell culture. Template DNA was extracted for 15 min using Extragen II (Tosoh, Tokyo, Japan) according to the manufacturer’s protocol [16]. DNA then was processed immediately for real-time PCR. HAdV primers and a molecular beacon (MB) probe were designed according to the hexon gene (GenBank: AB330084.1) encoding the viral structural protein. Sequences of primers and probe were those for the sense primer, 50 -CCCTGRTAKCCDATRTTGTA-30 ; and for the reverse primer, 50 -ACCTTYTAYCTBAAYCACAC-30 . The MB probe was labeled with a fluorescent reporter, 6-carboxyfluorescein (FAM) at the 50 -end and with black hole quencher 1 (BHQ-1) at the 30 -end; the probe sequence was 50 (FAM)-GCTGCCGAACCAGTCYTTKGTCATGTTRCA. GGCAGC-(BHQ-1)-30 . To achieve coverage of the main HAdV types, primers and probe were designed to detect 12 types: 1 to 8, 11, 19, 37, and 41. These detectable types were evaluated using clinical isolates. Real-time PCR was performed using an Agilent Technologies Stratagene Mx3000P (Agilent Technologies, La Jolla, CA). The reaction mixture consisted of 25 mL of iQ Multiplex Powermix (BioRad Laboratories, Hercules, CA) and 0.15 mL of primers and MB probe solution (final concentrations, both 300 nM). The final volume of the mixture was adjusted to 48 mL by addition of DNase- and RNase-free H2O (Nacalai Tesque, Inc., Kyoto, Japan). Finally, 2 mL of sample DNA was added to a tube kept on ice that contained the reaction mixture. Amplification was initiated at 95 C for 30 s, followed by 40 cycles of PCR reaction at 95 C for 15 s, at 50 C for 30 s, and at 75 C for 20 s, successively. Real-time PCR of this study was finished within 1.5 h. PCR sensitivity represented about 10 copies of HAdV DNA in per reaction mixture. 2.3. Rapid diagnostic tests The BD Veritor System Adeno kit is a rapid IC assay for direct qualitative detection of adenoviral antigens from clinical samples such as throat swabs, producing a result in 10 min. Results for HAdV were determined using the BD Veritor System Reader (Becton,
Dickinson and Company) not visual judgment. The limit of detection differed by serotype, ranging from 3.16 101 to 1.26 105 50% tissue culture infective dose (TCID50)/mL. For serotype 1, HAdV can be detected at 9.2 102 TCID50/mL. On the other hand, the BD Adeno Examan kit [20], commonly used for rapid identification of HAdV in Japan, can detect HAdV at about 5 102 viral particles per reaction, results is obtained within 15 min. Detectable HAdV serotypes were increased to 22 with BD Veritor System Adeno (1e8, 11, 13 to 16, 18 to 21, 26, 31, 37, 40, and 41), compared with 11 serotypes for BD Adeno Examan (1e8, 11, 19, and 37). 2.4. Adenovirus isolation and serotyping All samples (150 mL) were inoculated onto confluent monolayers of the A549 cell line. Inoculated cells were maintained in minimal essential medium supplemented with 1% fetal bovine serum (Cansera International Inc., Toronto, ON, Canada). Cell cultures were inspected for cytopathic effect (CPE) for 3e4 weeks. In the neutralization test for serotyping the supernatant from infected cells was diluted 100-fold, and then mixed with equal amounts of type-specific antiserum (5U). After incubation for 1 h, the mixture was inoculated in to the A549 cell line. HAdV type was identified by inhibition of CPE by each antiserum after during incubation for 1 week. 3. Results 3.1. Clinical findings and diseases Throat swab samples from 146 patients were evaluated. Patients were diagnosed with pharyngitis (n ¼ 67, 45.9%), tonsillitis (n ¼ 45, 30.8%), PCF (n ¼ 26, 17.8%), upper respiratory tract infection (URTI) (n ¼ 6, 4.1%), conjunctivitis (n ¼ 1, 0.7%), or bronchitis (n ¼ 1, 0.7%). Thirty-one of the patients (21.2%) had more than one disease (conjunctivitis, otitis media, bronchitis, pneumonia, gastroenteritis, exanthems, or influenza). Clinical findings were included fever in 144 patients (98.6%), pharyngeal pain in 49 (33.6%), conjunctivitis in 35 (24.0%), and tongue fur in 32 (21.9%). 3.2. Serotypes of HAdV isolates Table 1 shows relationships between diseases and serotypes of HAdV isolates. HAdV were isolated from 65 patients with pharyngitis (n ¼ 21), tonsillitis (27), PCF (n ¼ 15), and URTI (n ¼ 2) by cell culture. Serotype 3 (n ¼ 41; 63.1%) predominated among isolates, followed in order by serotype 2 (n ¼ 12; 18.5%), serotype 1 (n ¼ 6; Table 1 Relationship between disease and HAdV serotype determined using cell culture. Serotype
HAdV HAdV HAdV HAdV HAdV Total a b c
1 2 3 4 5
No. of positivesa
6 12 41 2 4 65
Clinical diagnosis (%) Pharyngitis
Tonsillitis
PCFb
URTIc
0 (0.0) 5 (41.7) 14 (34.1) 1 (50.0) 1 (25.0) 21
6 (100) 5 (41.7) 13 (31.7) 0 (0.0) 3 (75.0) 27
0 (0.0) 0 (0.0) 14 (34.1) 1 (50.0) 0 (0.0) 15
0 2 0 0 0 2
By cell culture. PCF; pharyngoconjunctival fever. URTI; upper respiratory tract infection.
(0.0) (16.7) (0.0) (0.0) (0.0)
M. Morozumi et al. / J Infect Chemother 20 (2014) 303e306 Table 2 Comparison of cell culture results with BD Veritor System Adeno, BD Adeno Examan, and real-time PCR. Serotypes
HAdV HAdV HAdV HAdV HAdV Total
1 2 3 4 5
No. of positives by cell culture
No. of patients (%) BD Veritor System Adeno
BD Adeno Examan
Real-time PCR
6 12 41 2 4 65
6 10 38 2 4 60
6 11 40 2 4 63
6 10 41 2 4 63
(100) (83.3) (92.7) (100) (100) (92.3)
(100) (91.7) (97.6) (100) (100) (96.9)
(100) (83.3) (100) (100) (100) (96.9)
9.2%), serotype 5 (n ¼ 4; 6.2%), and serotype 4 (n ¼ 2; 3.1%). These serotypes were assessed by all methods included in this study. 3.3. Comparison of culture results with IC assay kits, and real-time PCR Table 2 compares results from cell culture with those from IC assay kits, and real-time PCR. Of the 65 positive isolates, 63 (96.9%) were identified by BD Adeno Examan and real-time PCR, while 60 (92.3%) were identified by BD Veritor System Adeno. Positivity for serotype 2 was slightly less frequent than other types using IC assay kits or real-time PCR. 3.4. Sensitivity and specificity of the IC assay kits, and real-time PCR Sensitivities and specificities of IC assay kits, and real-time PCR determined according to results of cell culture are shown in Table 3. Of the 65 positive isolates, 3 were excluded from the analysis of BD Veritor System Adeno, because results with the BD Veritor System Reader were inconclusive. Relative sensitivity and specificity of BD Veritor System Adeno, BD Adeno Examan, and real-time PCR were 93.8% and 98.7%, 96.9% and 93.8%, and 96.9% and 100%, respectively. Positive predictive and negative predictive values for the 3 methods were 98.4% and 95.1%, 92.6% and 97.4%, and 100% and 97.6%, respectively. BD Adeno Examan (n ¼ 5; 6.2%) and BD Veritor System Adeno (n ¼ 1; 1.3%) each showed some false positive. Real-time PCR showed highest specificity, displaying complete correspondence to cell culture results. Efficiency of these methods was excellent: 96.5% for BD Veritor System Adeno, 95.2% for BD Adeno Examan, and 98.6% for real-time PCR. 4. Discussion Diagnosis of adenoviral infections usually is based on clinical findings and commercially available rapid diagnostic kits based on IC or ELISA [11e14]. Rapid diagnostic kits have proven to be a useful method for bedside diagnosis or outpatient care of HAdV infections. HAdV infection is difficult to diagnose by the presence of symptoms
305
alone. HAdV infections can be associated with high white blood cell counts (WBC) and C-reactive protein (CRP) concentrations [16,21,22]. Accordingly, adenoviral infections and bacterial infections can be difficult to differentiate using clinical laboratory values. Rapid identification of adenoviral infections in pediatric patients often avoids unnecessary of prescriptions antibiotics and can prevent or contain HAdV-related outbreaks. Both cell culture and real-time PCR also are used for identification of adenoviral infections [15e19]. Although culture for HAdV usually requires a week, real-time PCR provides results with high sensitivity within a few hours, additionally, it permits quantitative determination of adenoviral loads in clinical samples. Identifying HAdV by real-time PCR is very helpful in the clinical setting, especially during epidemics in place of time-consuming culture. Recently, real-time PCR method of Luminex xTAG REV Fast has been approved by FDA [23]. In addition, some new methods, such as LightCycler [24], LAMP [19], and TaqMan [24e26] have been developed to identify HAdV. In the present study, we compared the results of cell culture, commercially available IC assay kits, and real-time PCR with MB probe for throat swab samples from pediatric patients. As previous reports (11e14), real-time PCR showed highest sensitivity and specificity, IC kit also showed sufficient sensitivity and specificity for diagnosis. In this study, the sensitivity and specificity of real-time PCR (96.9% and 100%) was greater than that of IC assay kits (93.8% and 98.7% for BD Veritor System Adeno kit, and 96.9% and 93.8% for BD Adeno Examan kit). As determined by the BD Veritor System Reader, the BD Veritor System Adeno kit showed fewer false positives (1.3%) than the BD Adeno Examan kit (6.2%). Serotypes 1 to 5 were detected in this study, representing known causes of pharyngitis and tonsillitis [3,7]. Several studies have found that in Japan, severe conjunctivitis such as EKC, PCF, and nonspecific follicular conjunctivitis is caused predominantly by serotypes 3, 4, 8, 11, 19, and 37 [27e29] and serotypes 40 and 41 previously were prevalent among pediatric patients with enteritis or acute gastroenteritis [30,31]. The BD Veritor System Adeno kit includes these epidemic serotypes, it can detect more HAdV serotypes than another IC kit or real-time PCR. Though real-time PCR can detect HAdV with high sensitivity and specificity, dedicated equipment and reagents are required. Using a rapid diagnostic kit with IC, HAdV can be detected easily by clinicians within 15 min appropriate for clinical use. Results with IC kits provide a sufficient basis for clinicians to initiate antiviral therapy and/or implement infection-control measures. The rapid diagnostic kits and real-time PCR for HAdV can minimize the risk of spreading HAdV infections and limiting epidemics and these methods are useful for diagnosing HAdV infections in pediatric patients. Conflict of interest The authors declare that they have no conflict of interest.
Table 3 Sensitivities and specificities of BD Veritor System Adeno, BD Adeno Examan, and real-time PCR, compared with cell culture. Method
Sensitivity (%) 95% CI
Specificity (%) 95% CI
Positive predictive value 95% CI
Negative predictive value 95% CI
BD Veritor System Adeno
60/64 (93.8) 84.8e98.3 63/65 (96.9) 89.3e99.6 63/65 (96.9) 89.3e99.6
78/79 (98.7) 93.1e100 76/81 (93.8) 86.2e98.0 81/81 (100) 95.5e100
60/61 (98.4) 91.2e100 63/68 (92.6) 83.7e97.6 63/63 (100) 94.3e100
78/82 (95.1) 88.0e98.7 76/78 (97.4) 91.0e99.7 81/83 (97.6) 91.6e99.7
BD Adeno Examan Real-time PCR
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Acknowledgements We thank the members who participated in this study; Drs. Masataka Ichikawa (Ichikawa Children’s Clinic), Shuko Okano (Okano Children’s Clinic), Osamu Kakuta (Medical Corporation Tomosatokai Kakuta Clinic), Yasuo Kondo (Kondo Children’s Clinic), Toyoaki Nakayama (Nakayama Clinic), Shuichi Nishimura (Nishimura Clinic), Toshiyuki Hikita (Hikita Pediatric Clinic), Yosuke Mori (Mori Children’s Clinic), and Masahiko Yamazaki (Zama Children’s Clinic). We also thank S. Masuyoshi of Kitasato Institute for Life Sciences, Kitasato University for assistance with this study. References [1] Gardner PS. Virus infections and respiratory disease of childhood. Arch Dis Child 1968;43:629e45. [2] Lynch 3rd JP, Fishbein M, Echavarria M. Adenovirus. Semin Respir Crit Care Med 2011;32:494e511. [3] Robinson C, Echavarria M. Adenoviruses. In: Murray P, Baron EJ, Jorgensen JH, Landry ML, Pfaller MA, editors. Manual of clinical microbiology. 9th ed. Washington DC: American Society for Microbiology; 2007. pp. 1589e600. [4] de Jong JC, Osterhaus AD, Jones MS, Harrach B. Human adenovirus type 52: a type 41 in disguise? J Virol 2008;82:3809e10. [5] Jones 2nd MS, Harrach B, Ganac RD, Gozum MM, Dela Cruz WP, Riedel B, et al. New adenovirus species found in a patient presenting with gastroenteritis. J Virol 2007;81:5978e84. [6] Robinson CM, Singh G, Henquell C, Walsh MP, Peigue-Lafeuille H, Seto D, et al. Computational analysis and identification of an emergent human adenovirus pathogen implicated in a respiratory fatality. Virology 2011;409:141e7. [7] Kunz AN, Ottolini M. The role of adenovirus in respiratory tract infections. Curr Infect Dis Rep 2010;12:81e7. [8] Takimoto S, Grandien M, Ishida MA, Pereira MS, Paiva TM, Ishimaru T, et al. Comparison of enzyme-linked immunosorbent assay, indirect immunofluorescence assay, and virus isolation for detection of respiratory viruses in nasopharyngeal secretions. J Clin Microbiol 1991;29:470e4. [9] Ray CG, Minnich LL. Efficiency of immunofluorescence for rapid detection of common respiratory viruses. J Clin Microbiol 1987;25:355e7. [10] Shetty AK, Treynor E, Hill DW, Gutierrez KM, Warford A, Baron EJ. Comparison of conventional viral cultures with direct fluorescent antibody stains for diagnosis of community-acquired respiratory virus infections in hospitalized children. Pediatr Infect Dis J 2003;22:789e94. [11] Fujimoto T, Okafuji T, Okafuji T, Ito M, Nukuzuma S, Chikahira M, et al. Evaluation of a bedside immunochromatographic test for detection of adenovirus in respiratory samples, by comparison to virus isolation, PCR, and real-time PCR. J Clin Microbiol 2004;42:5489e92. [12] Goto E. Performance evaluation of detecting adenovirus by using rapid diagnostic kits among Japanese people. J Infect Chemother 2012;18:361e9. [13] Levent F, Greer JM, Snider M, Demmler-Harrison GJ. Performance of a new immunochromatographic assay for detection of adenoviruses in children. J Clin Virol 2009;44:173e5. [14] Rovida F, Campanini G, Sarasini A, Adzasehoun KM, Piralla A, Baldanti F. Comparison of immunologic and molecular assays for the diagnosis of gastrointestinal viral infections. Diagn Microbiol Infect Dis 2013;75:110e1.
[15] Buckwalter SP, Teo R, Espy MJ, Sloan LM, Smith TF, Pritt BS. Real-time qualitative PCR for 57 human adenovirus types from multiple specimen sources. J Clin Microbiol 2012;50:766e71. [16] Hamano-Hasegawa K, Morozumi M, Nakayama E, Chiba N, Murayama SY, Takayanagi R, et al. Comprehensive detection of causative pathogens using real-time PCR to diagnose pediatric community-acquired pneumonia. J Infect Chemother 2008;14:424e32. [17] Jeulin H, Salmon A, Bordigoni P, Venard V. Comparison of in-house real-time quantitative PCR to the adenovirus R-Gene kit for determination of adenovirus load in clinical samples. J Clin Microbiol 2010;483:132e7. s¸anu D, Zapucioiu C, Matei D, et al. The [18] Tecu C, Mihai ME, Alexandrescu VI, Ora adenoviral infections in children admitted to hospital with pneumonia, acute bronchiolitis or respiratory viral infections. Roum Arch Microbiol Immunol 2012;71:24e8. [19] Wakabayashi T, Yamashita R, Kakita T, Kakita M, Oshika T. Rapid and sensitive diagnosis of adenoviral keratoconjunctivitis by loop-mediated isothermal amplification (LAMP) method. Curr Eye Res 2004;29:219e24. [20] Shimizu H, Ishimaru Y, Fujimoto T. Evaluation of immunochromatographic detection kit using adenovirus PteAu colloid. Kansenshogaku Zasshi 2009;83: 64e5. [21] Peltola V, Mertsola J, Ruuskanen O. Comparison of total white blood cell count and serum C-reactive protein levels in confirmed bacterial and viral infections. J Pediatr 2006;149:721e4. [22] Tabain I, Ljubin-Sternak S, Cepin-Bogovi c J, Markovinovi c L, Knezovi c I, Mlinari c-Galinovi c G. Adenovirus respiratory infections in hospitalized children: clinical findings in relation to species and serotypes. Pediatr Infect Dis J 2012;31:680e4. [23] Pabbaraju K, Wong S, Tokaryk KL, Fonseca K, Drews SJ. Comparison of the Luminex xTAG respiratory viral panel with xTAG respiratory viral panel fast for diagnosis of respiratory virus infections. J Clin Microbiol 2011;49: 1738e44. [24] Jones MS, Hudson NR, Gibbins C, Fischer SL. Evaluation of type-specific realtime PCR assays using the LightCycler and J.B.A.I.D.S. for detection of adenoviruses in species HAdV-C. PLoS One 2011;6:e26862. [25] Feeney SA, Armstrong VJ, Mitchell SJ, Crawford L, McCaughey C, Coyle PV. Development and clinical validation of multiplex TaqManÒ assays for rapid diagnosis of viral gastroenteritis. J Med Virol 2011;83:1650e6. [26] Liu P, Herzegh O, Fernandez M, Hooper S, Shu W, Sobolik J, et al. Assessment of human adenovirus removal by qPCR in an advanced water reclamation plant in Georgia, USA. J Appl Microbiol 2013;15:310e8. [27] Yamadera S, Yamashita K, Akatsuka M, Kato N, Hashido M, Inouye S, et al. Adenovirus surveillance, 1982-1993, Japan. A report of the National Epidemiological surveillance of Infectious Agents in Japan. Jpn J Med Sci Biol 1995;48:199e210. [28] Adhikary AK, Banik U, Okabe N, Fujimoto T. Molecular characterization of human adenovirus type 8 (HAdV-8), including a novel genome type detected in Japan. Jpn J Infect Dis 2010;64:493e8. [29] Saitoh-Inagawa W, Oshima A, Aoki K, Itoh N, Isobe K, Uchio E, et al. Rapid diagnosis of adenoviral conjunctivitis by PCR restriction fragment length polymorphism analysis. J Clin Microbiol 1996;34:2113e6. [30] Li L, Phan TG, Nguyen TA, Kim KS, Seo JK, Shimizu H, et al. Molecular epidemiology of adenovirus infection among pediatric population with diarrhea in Asia. Microbiol Immunol 2005;49:121e8. [31] Shinozaki T, Araki K, Fujita Y, Kobayashi M, Tajima T, Abe T. Epidemiology of enteric adenoviruses 40 and 41 in acute gastroenteritis in infants and young children in the Tokyo area. Scand J Infect Dis 1991;23:543e7.
Contents lists available at ScienceDirect
Journal of Infection and Chemotherapy journal homepage: http://www.elsevier.com/locate/jic
Original article
Evaluation of new immunochromatographic assay kit for adenovirus detection in throat swab: Comparison with culture and real-time PCR results Miyuki Morozumi a, b, Hideaki Shimizu c, Yuki Matsushima c, Keiko Mitamura d, Takeshi Tajima e, Satoshi Iwata b, Kimiko Ubukata, PhD a, b, * a
Laboratory of Molecular Epidemiology for Infectious Agents, Kitasato Institute for Life Sciences, Kitasato University, Tokyo, Japan Department of Infectious Diseases, Keio University School of Medicine, Tokyo, Japan Division of Virology, Kawasaki City Institute for Public Health, Kanagawa, Japan d Department of Pediatrics, Eiju General Hospital, Tokyo, Japan e Department of Pediatrics, Hakujikai Memorial Hospital, Tokyo, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 September 2013 Received in revised form 10 January 2014 Accepted 14 January 2014
A new immunochromatographic (IC) assay kit, BD Veritor System Adeno was evaluated to comparing with commercial available kit, BD Adeno Examan, cell culture, and real-time PCR using throat swab samples. Specimens were collected from 146 pediatric patients between July 2011 and January 2012. Mean age of patients was 4 years (8 monthse15 years old). Patients were diagnosed with pharyngitis (n ¼ 67), tonsillitis (n ¼ 45), pharyngoconjunctival fever (n ¼ 26), upper respiratory tract infection (n ¼ 6), conjunctivitis (n ¼ 1), or bronchitis (n ¼ 1). Thirty-one of the patients (21.2%) had more than one disease. Among all samples, 61 (41.8%) were positive for adenovirus with BD Veritor System Adeno; 68 (46.6%) with BD Adeno Examan; 63 (43.2%) with real-time PCR; and 65 (44.5%) with cell culture. Serotype 3 (n ¼ 41; 63.1%) was predominant among the 65 adenovirus isolates, followed by serotype 2 (n ¼ 12; 18.5%), 1 (n ¼ 6; 9.2%), 5 (n ¼ 4; 6.2%), and 4 (n ¼ 2; 3.1%). Relative sensitivity and specificity of BD Veritor System Adeno, BD Adeno Examan, and real-time PCR were 93.8% and 98.7%, 96.9% and 93.8%, and 96.9% and 100%, respectively. Positive predictive and negative predictive values for these methods were 98.4% and 95.1%, 92.6% and 97.4%, and 100% and 97.6%, respectively. The sensitivity and specificity of real-time PCR was greater than that of IC assay kits. However, IC assay kits also showed high sensitivity and specificity appropriate for clinical use. Ó 2014, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Keywords: Adenovirus Rapid detection Immunochromatographic assay Real-time PCR Serotype
1. Introduction Human adenoviruses (HAdV) are major causes of respiratory tract infections [1e3]. The most common adenoviral infections are pneumonia, bronchitis, pharyngitis, tonsillitis, conjunctivitis, and pharyngoconjunctival fever (PCF). Currently, 7 HAdV species designated have been identified A to G. These species include more than 50 serotypes [4e6]. HAdV serotypes have been characterized according to illness caused: are serotypes 1 to 7 are related to upper * Corresponding author. Department of Infectious Diseases, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Tel.: þ81 3 3353 1211; fax: þ81 3 5363 3711. E-mail address: [email protected] (K. Ubukata).
respiratory tract infection; serotypes 3, 7, 14, and 21, to lower respiratory tract infection; serotypes 3, 4, and 7, to PCF; and serotypes 8, 19, and 37, to epidemic keratoconjunctivitis (EKC) [3,5,7]. Standard diagnostic methods for adenoviral infections include cell culture and antigen detection [8e10]. Although adenovirus can be isolated by cell culture considerable time is required. Commercially available rapid diagnostic kits using immunochromatography (IC) or enzyme-linked immunosorbent assay (ELISA) have been developed for early diagnosis of adenoviral infections [11e14]. Meanwhile, conventional PCR or real-time PCR can detect adenoviral DNA rapidly and also can obtain quantitative results with high sensitivity and specificity [15e19]. Early detection of HAdV is important for both guiding treatment and preventing spread of the infection.
http://dx.doi.org/10.1016/j.jiac.2014.01.005 1341-321X/Ó 2014, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
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To evaluate of a new rapid diagnostic kit, BD Veritor System Adeno, we compared the results of cell culture, IC assay kits (BD Veritor System Adeno; Becton and BD Adeno Examan; Dickinson and Company, Franklin Lakes, NJ), and real-time PCR using throat swab samples from pediatric patients. 2. Materials and methods 2.1. Patients and clinical samples Eleven medical institutions participated actively in this study from July 2011 to January 2012. All together, 146 clinical samples were collected from pediatric outpatients diagnosed with pharyngitis, tonsillitis, or conjunctivitis likely caused by HAdV based on clinical symptoms and findings such as fever (temperature exceeding 37.0 C), pharyngeal pain, tongue fur, and conjunctivitis. Throat swab samples were collected using universal transport media (Becton, Dickinson and Company) for cell culture. After informed consent was obtained from patients, parents, samples were collected by pediatricians. Mean age of patients was 4 years (8 monthse15 years old); 88 were male. 2.2. Real-time PCR to detect adenovirus Detection of HAdV DNA in specimens was carried out by realtime PCR. Clinical samples were centrifuged at 2000 g at 4 C for 10 min to collect virus attached to cells. One hundred and fifty microliter containing pellet after the supernatant discarded was mixed gently. The supernatant was used for cell culture. Template DNA was extracted for 15 min using Extragen II (Tosoh, Tokyo, Japan) according to the manufacturer’s protocol [16]. DNA then was processed immediately for real-time PCR. HAdV primers and a molecular beacon (MB) probe were designed according to the hexon gene (GenBank: AB330084.1) encoding the viral structural protein. Sequences of primers and probe were those for the sense primer, 50 -CCCTGRTAKCCDATRTTGTA-30 ; and for the reverse primer, 50 -ACCTTYTAYCTBAAYCACAC-30 . The MB probe was labeled with a fluorescent reporter, 6-carboxyfluorescein (FAM) at the 50 -end and with black hole quencher 1 (BHQ-1) at the 30 -end; the probe sequence was 50 (FAM)-GCTGCCGAACCAGTCYTTKGTCATGTTRCA. GGCAGC-(BHQ-1)-30 . To achieve coverage of the main HAdV types, primers and probe were designed to detect 12 types: 1 to 8, 11, 19, 37, and 41. These detectable types were evaluated using clinical isolates. Real-time PCR was performed using an Agilent Technologies Stratagene Mx3000P (Agilent Technologies, La Jolla, CA). The reaction mixture consisted of 25 mL of iQ Multiplex Powermix (BioRad Laboratories, Hercules, CA) and 0.15 mL of primers and MB probe solution (final concentrations, both 300 nM). The final volume of the mixture was adjusted to 48 mL by addition of DNase- and RNase-free H2O (Nacalai Tesque, Inc., Kyoto, Japan). Finally, 2 mL of sample DNA was added to a tube kept on ice that contained the reaction mixture. Amplification was initiated at 95 C for 30 s, followed by 40 cycles of PCR reaction at 95 C for 15 s, at 50 C for 30 s, and at 75 C for 20 s, successively. Real-time PCR of this study was finished within 1.5 h. PCR sensitivity represented about 10 copies of HAdV DNA in per reaction mixture. 2.3. Rapid diagnostic tests The BD Veritor System Adeno kit is a rapid IC assay for direct qualitative detection of adenoviral antigens from clinical samples such as throat swabs, producing a result in 10 min. Results for HAdV were determined using the BD Veritor System Reader (Becton,
Dickinson and Company) not visual judgment. The limit of detection differed by serotype, ranging from 3.16 101 to 1.26 105 50% tissue culture infective dose (TCID50)/mL. For serotype 1, HAdV can be detected at 9.2 102 TCID50/mL. On the other hand, the BD Adeno Examan kit [20], commonly used for rapid identification of HAdV in Japan, can detect HAdV at about 5 102 viral particles per reaction, results is obtained within 15 min. Detectable HAdV serotypes were increased to 22 with BD Veritor System Adeno (1e8, 11, 13 to 16, 18 to 21, 26, 31, 37, 40, and 41), compared with 11 serotypes for BD Adeno Examan (1e8, 11, 19, and 37). 2.4. Adenovirus isolation and serotyping All samples (150 mL) were inoculated onto confluent monolayers of the A549 cell line. Inoculated cells were maintained in minimal essential medium supplemented with 1% fetal bovine serum (Cansera International Inc., Toronto, ON, Canada). Cell cultures were inspected for cytopathic effect (CPE) for 3e4 weeks. In the neutralization test for serotyping the supernatant from infected cells was diluted 100-fold, and then mixed with equal amounts of type-specific antiserum (5U). After incubation for 1 h, the mixture was inoculated in to the A549 cell line. HAdV type was identified by inhibition of CPE by each antiserum after during incubation for 1 week. 3. Results 3.1. Clinical findings and diseases Throat swab samples from 146 patients were evaluated. Patients were diagnosed with pharyngitis (n ¼ 67, 45.9%), tonsillitis (n ¼ 45, 30.8%), PCF (n ¼ 26, 17.8%), upper respiratory tract infection (URTI) (n ¼ 6, 4.1%), conjunctivitis (n ¼ 1, 0.7%), or bronchitis (n ¼ 1, 0.7%). Thirty-one of the patients (21.2%) had more than one disease (conjunctivitis, otitis media, bronchitis, pneumonia, gastroenteritis, exanthems, or influenza). Clinical findings were included fever in 144 patients (98.6%), pharyngeal pain in 49 (33.6%), conjunctivitis in 35 (24.0%), and tongue fur in 32 (21.9%). 3.2. Serotypes of HAdV isolates Table 1 shows relationships between diseases and serotypes of HAdV isolates. HAdV were isolated from 65 patients with pharyngitis (n ¼ 21), tonsillitis (27), PCF (n ¼ 15), and URTI (n ¼ 2) by cell culture. Serotype 3 (n ¼ 41; 63.1%) predominated among isolates, followed in order by serotype 2 (n ¼ 12; 18.5%), serotype 1 (n ¼ 6; Table 1 Relationship between disease and HAdV serotype determined using cell culture. Serotype
HAdV HAdV HAdV HAdV HAdV Total a b c
1 2 3 4 5
No. of positivesa
6 12 41 2 4 65
Clinical diagnosis (%) Pharyngitis
Tonsillitis
PCFb
URTIc
0 (0.0) 5 (41.7) 14 (34.1) 1 (50.0) 1 (25.0) 21
6 (100) 5 (41.7) 13 (31.7) 0 (0.0) 3 (75.0) 27
0 (0.0) 0 (0.0) 14 (34.1) 1 (50.0) 0 (0.0) 15
0 2 0 0 0 2
By cell culture. PCF; pharyngoconjunctival fever. URTI; upper respiratory tract infection.
(0.0) (16.7) (0.0) (0.0) (0.0)
M. Morozumi et al. / J Infect Chemother 20 (2014) 303e306 Table 2 Comparison of cell culture results with BD Veritor System Adeno, BD Adeno Examan, and real-time PCR. Serotypes
HAdV HAdV HAdV HAdV HAdV Total
1 2 3 4 5
No. of positives by cell culture
No. of patients (%) BD Veritor System Adeno
BD Adeno Examan
Real-time PCR
6 12 41 2 4 65
6 10 38 2 4 60
6 11 40 2 4 63
6 10 41 2 4 63
(100) (83.3) (92.7) (100) (100) (92.3)
(100) (91.7) (97.6) (100) (100) (96.9)
(100) (83.3) (100) (100) (100) (96.9)
9.2%), serotype 5 (n ¼ 4; 6.2%), and serotype 4 (n ¼ 2; 3.1%). These serotypes were assessed by all methods included in this study. 3.3. Comparison of culture results with IC assay kits, and real-time PCR Table 2 compares results from cell culture with those from IC assay kits, and real-time PCR. Of the 65 positive isolates, 63 (96.9%) were identified by BD Adeno Examan and real-time PCR, while 60 (92.3%) were identified by BD Veritor System Adeno. Positivity for serotype 2 was slightly less frequent than other types using IC assay kits or real-time PCR. 3.4. Sensitivity and specificity of the IC assay kits, and real-time PCR Sensitivities and specificities of IC assay kits, and real-time PCR determined according to results of cell culture are shown in Table 3. Of the 65 positive isolates, 3 were excluded from the analysis of BD Veritor System Adeno, because results with the BD Veritor System Reader were inconclusive. Relative sensitivity and specificity of BD Veritor System Adeno, BD Adeno Examan, and real-time PCR were 93.8% and 98.7%, 96.9% and 93.8%, and 96.9% and 100%, respectively. Positive predictive and negative predictive values for the 3 methods were 98.4% and 95.1%, 92.6% and 97.4%, and 100% and 97.6%, respectively. BD Adeno Examan (n ¼ 5; 6.2%) and BD Veritor System Adeno (n ¼ 1; 1.3%) each showed some false positive. Real-time PCR showed highest specificity, displaying complete correspondence to cell culture results. Efficiency of these methods was excellent: 96.5% for BD Veritor System Adeno, 95.2% for BD Adeno Examan, and 98.6% for real-time PCR. 4. Discussion Diagnosis of adenoviral infections usually is based on clinical findings and commercially available rapid diagnostic kits based on IC or ELISA [11e14]. Rapid diagnostic kits have proven to be a useful method for bedside diagnosis or outpatient care of HAdV infections. HAdV infection is difficult to diagnose by the presence of symptoms
305
alone. HAdV infections can be associated with high white blood cell counts (WBC) and C-reactive protein (CRP) concentrations [16,21,22]. Accordingly, adenoviral infections and bacterial infections can be difficult to differentiate using clinical laboratory values. Rapid identification of adenoviral infections in pediatric patients often avoids unnecessary of prescriptions antibiotics and can prevent or contain HAdV-related outbreaks. Both cell culture and real-time PCR also are used for identification of adenoviral infections [15e19]. Although culture for HAdV usually requires a week, real-time PCR provides results with high sensitivity within a few hours, additionally, it permits quantitative determination of adenoviral loads in clinical samples. Identifying HAdV by real-time PCR is very helpful in the clinical setting, especially during epidemics in place of time-consuming culture. Recently, real-time PCR method of Luminex xTAG REV Fast has been approved by FDA [23]. In addition, some new methods, such as LightCycler [24], LAMP [19], and TaqMan [24e26] have been developed to identify HAdV. In the present study, we compared the results of cell culture, commercially available IC assay kits, and real-time PCR with MB probe for throat swab samples from pediatric patients. As previous reports (11e14), real-time PCR showed highest sensitivity and specificity, IC kit also showed sufficient sensitivity and specificity for diagnosis. In this study, the sensitivity and specificity of real-time PCR (96.9% and 100%) was greater than that of IC assay kits (93.8% and 98.7% for BD Veritor System Adeno kit, and 96.9% and 93.8% for BD Adeno Examan kit). As determined by the BD Veritor System Reader, the BD Veritor System Adeno kit showed fewer false positives (1.3%) than the BD Adeno Examan kit (6.2%). Serotypes 1 to 5 were detected in this study, representing known causes of pharyngitis and tonsillitis [3,7]. Several studies have found that in Japan, severe conjunctivitis such as EKC, PCF, and nonspecific follicular conjunctivitis is caused predominantly by serotypes 3, 4, 8, 11, 19, and 37 [27e29] and serotypes 40 and 41 previously were prevalent among pediatric patients with enteritis or acute gastroenteritis [30,31]. The BD Veritor System Adeno kit includes these epidemic serotypes, it can detect more HAdV serotypes than another IC kit or real-time PCR. Though real-time PCR can detect HAdV with high sensitivity and specificity, dedicated equipment and reagents are required. Using a rapid diagnostic kit with IC, HAdV can be detected easily by clinicians within 15 min appropriate for clinical use. Results with IC kits provide a sufficient basis for clinicians to initiate antiviral therapy and/or implement infection-control measures. The rapid diagnostic kits and real-time PCR for HAdV can minimize the risk of spreading HAdV infections and limiting epidemics and these methods are useful for diagnosing HAdV infections in pediatric patients. Conflict of interest The authors declare that they have no conflict of interest.
Table 3 Sensitivities and specificities of BD Veritor System Adeno, BD Adeno Examan, and real-time PCR, compared with cell culture. Method
Sensitivity (%) 95% CI
Specificity (%) 95% CI
Positive predictive value 95% CI
Negative predictive value 95% CI
BD Veritor System Adeno
60/64 (93.8) 84.8e98.3 63/65 (96.9) 89.3e99.6 63/65 (96.9) 89.3e99.6
78/79 (98.7) 93.1e100 76/81 (93.8) 86.2e98.0 81/81 (100) 95.5e100
60/61 (98.4) 91.2e100 63/68 (92.6) 83.7e97.6 63/63 (100) 94.3e100
78/82 (95.1) 88.0e98.7 76/78 (97.4) 91.0e99.7 81/83 (97.6) 91.6e99.7
BD Adeno Examan Real-time PCR
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