Detection of Norovirus Antigens from Recombinant Virus-Like Particles
http://www.100md.com
《微生物临床杂志》
Department of Developmental Medical Sciences, School of International Health, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
Nippon Becton Dickinson Company, Tokyo, Japan
Institüt fur Physiologische Chemie, Abteilung Angewandte Molekularbilogie, Johannes Gutenberg-Universitat, Mainz, Germany
ABSTRACT
The commercial norovirus enzyme-linked immunosorbent assay kit was evaluated for its reactivity to recombinant virus-like particles and the detection of natural viruses from stool samples of Japanese infants and children with sporadic acute gastroenteritis compared to reverse transcription-PCR. The kit had a sensitivity of 76.3% and a specificity of 94.9%. Our results clearly indicated that the kit allows the detection of the most prevalent genotype, GII/4. In order to increase the sensitivity of the kit, the reactivity with norovirus of GII/3 and GII/6 genotypes needs to be improved.
TEXT
Norovirus (NoV) is one of the leading etiologic agents of nonbacterial sporadic acute gastroenteritis (AGE) in infants and children, and outbreaks of this infection may be due to contaminated water or food. At present, the reverse transcription-PCR (RT-PCR) assay is widely used to detect NoV in diarrheal stool samples. The development of immunological methods to detect NoV has been delayed due to the lack of viruses in cell culture and to diverse genotypes with distinct antigenicities. NoVs are currently divided into five genogroups, and most human NoV strains belong to two genogroups: genogroup I (GI) and genogroup II (GII). Furthermore, each genogroup contains at least 15 and 18 genotypes, respectively (13). RT-PCR is an expensive and complicated technique, and its use requires special equipment and skills. Thus, a faster and simpler method is needed. At present, three commercial enzyme-linked immunosorbent assay (ELISA) kits are available, the IDEIA NLV kit from Dako Cytomation, Ltd. (Ely, United Kingdom), the SRSV(II)-AD kit from Denka Seiken Co., Ltd. (Tokyo, Japan), and the RIDASCREEN norovirus (R-Biopharm AG, Darmstadt, Germany). According to previous evaluations of the ELISA kits, the first two kits cannot effectively replace RT-PCR for NoV detection due to their low sensitivities and/or specificities (1, 3, 19). To date, the RIDASCREEN norovirus assay has only been evaluated by one Australian group using outbreak specimens (2). No research has hitherto been conducted using recombinant virus-like particles (rVLPs) and sporadic stool samples.
Therefore, using the RIDASCREEN norovirus ELISA kit, we set out to measure the reactivity of 16 kinds of rVLPs, to detect the presence of NoV in fecal samples from infants and children with sporadic AGE in Japan, and to compare the sensitivity and specificity data with those obtained with the RT-PCR.
We previously expressed one rVLP (strain 1207, GII/4) (14). The other 15 rVLPs were prepared from NoV isolated from stool samples among infants and children with diarrhea between 1995 and 2003 in Japan. The genotypic classification of these NoV was performed based on the method described by Kageyama et al. (6). These are genotypes 1 (strain 4656), 3 (strain 3634), 4 (strain 2876), 8 (strain 3006), and 11 (strain 2258) in genogroup I and genotypes 1 (strain 3101), 2 (strain 2840), 3 (strain 3229), 5 (strain 3611), 6 (strain 3612), 7 (strain 419), 12 (strain 2087), 13 (strain 3385), 14 (strain 2468), and 15 (strain 3625) in genogroup II. The production of recombinant bacmids was performed using the baculovirus expression system with Gateway Technology (Invitrogen Japan, Tokyo) and the transfection of bacmids into insect cells, as well as the purification of rVLPs, was performed according to the method of Hansman et al. (5). We used two sense primers: attB1NVGI (GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT CGA AGG AGA TAG AAC CAT GAT GAT GGC GTC TAA GG) for GI strains and attB1NVGII (GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT CGA AGG AGA TAG AAC CAT GAA GAT GGC GTC GAA TGA) for GII strains. Purified rVLPs from the cultured supernatants of the inset cells were examined for particle formation by electron microscopy. Protein concentration of each rVLP was measured by BCA Coomassie protein assay (Pierce Biotechnology, Inc., Rockford, IL), and 150 μg/ml was prepared as stock solutions. The assays were started from 10 μg/ml as the highest concentration.
The rVLPs stock solutions were serially threefold diluted with the sample dilution buffer in the the RIDASCREEN norovirus ELISA kit and used to determine the minimal concentration of each rVLP for detection by ELISA according to the manufacturer's manual. All of the assays except that for GI/3 were done with kits of the same lot number. In the manual, the cutoff value is calculated as an absorbance value of negative control plus 0.15. Values that are 10% above or below the cutoff value are considered to be in the gray zone and therefore need to be examined again. In view of this, the theoretical minimal detectable concentration of each rVLP was determined as a calculated value which gave an absorbance value that was 10% above the cutoff value in each assay. Each assay was conducted in triplicate, and the experiment for each rVLP was repeated three to six times.
Five hundred and three stool samples were collected from infants and children with AGE who visited six pediatric clinics in Sapporo, Tokyo, Maizuru, and Osaka, Japan, from July 2004 to March 2005. All of the stool samples were stored at –30°C until testing. Watery stool samples were diluted 1:2 with phosphate-buffered saline (PBS), and hard stool samples were suspended to 1:5 with PBS. The suspensions were clarified by centrifugation at 10,000 x g for 15 min. The supernatants were diluted to 1:3 with the sample dilution buffer of the kit and used for the assay. The positives or negatives of the samples were determined as mentioned above.
Ten percent stool suspensions of 503 samples were prepared with PBS from the same aliquots for ELISA, and viral RNA was extracted by the QIA amp viral RNA mini kit (QIAGEN, Tokyo, Japan). The detection of NoV (GI and GII), astrovirus, sapovirus, rotavirus, and adenovirus was performed by two sets of multiplex PCR (21, 22). NoV-negative samples were examined by using two sets of monoplex PCRs, for NoV GI and GII. Twenty samples were further assayed by seminested PCR using a primer set, which were G2SKF and G2SKR for NoV GII (9). The genotypes of NoV were determined according to the method of Phan et al. (16).
The minimal detectable sensitivity is indicated in Table 1. The kit could detect GI/1 and GII/4 rVLPs at concentrations of <0.01 μg/ml. rVLPs of GI/3, GI/8, GI/11, GII/1, GII/2, and GII/12 were detectable within a range between 0.04 and 1 μg/ml. On the other hand, rVLPs of GI/4, GII/3, GII/5, GII/6, GII/14, and GII/15 were detected at more than 1 μg/ml. In cases where the assays for GI/4, GII/3, GII/6, GII/14, and GII/15 could not be detected at concentrations of <10 μg/ml and theoretical detectable concentrations could not be calculated, minimal concentrations given by the assay have been indicated (Table 1). GII/6 rVLP could be detected once at the highest concentration, 10 μg/ml. Two rVLPs of GII/7 and GII/13 could not be detected at a concentration of <10 μg/ml.
NoV in stool samples collected from sporadic cases in Japan was examined using both the ELISA kit and the RT-PCR, and the kit was evaluated based on the RT-PCR (Table 2). The calculated percent sensitivity, specificity, and agreement were 76.3, 94.9, and 90.7%, respectively. Twenty samples were determined to be positive by the kit but negative by the RT-PCR. These samples became positive when tested by the seminested PCR using NoV GII-specific primer pair. A total of 27 samples were positive with the RT-PCR but negative with the kit. The genotypes of 134 positive stool samples recorded by the RT-PCR were identified by using the clustering determined by Kageyama et al. (6) (Table 3). The genotypes of kit-positive, PCR-positive samples were 1 GI/1, 3 GII/3, 82 GII/4, and 1 GII/6, and the sensitivities of GI/1, GII/3, GII/4, and GII/6 were 50, 23.1, 85.4, and 33.3%, respectively. The low sensitivities of GII/3 and GII/6 were comparable to the results for the rVLPs. RT-multiplex PCR detected four other species of viruses in 503 stool samples. These were 7 group A rotavirus, 27 adenovirus, 30 sapovirus, and 1 astrovirus samples, and the stool samples containing these viruses were determined to be negative by ELISA. Furthermore, multiplex-PCR indicated that 8 of 112 NoV GII-positive samples were mixed infected with other viruses (5 sapovirus, 2 group A rotavirus, and 1 adenovirus).
Some studies showed that the strains belonging to GII/4 cluster were most predominant not only in stool samples from sporadic cases involving infants and children but also from the outbreaks (8, 10, 12, 15, 16, 19). On the other hand, it was found that various genotypes of NoV strains were detected in the outbreak cases, and there were no predominant genotypes in outbreak strains (20). Furthermore, a change in the distribution of NoV genotypes in the sporadic cases and the emergence of recombinant viruses has been reported (7, 11, 17, 18).
The ELISA kit could detect two kinds of rVLPs (GI/1 and GII/4) with a high sensitivity. Meanwhile, the GII/3 and GII/6 rVLPs formed a group that was responsive at higher concentrations. A total of 23.1% of the stool samples containing GII/3 NoV, and 33.3% of the samples with the GII/6 genotype could be effectively examined by the kit. NoV genotypes with low reactivity levels in the stool samples could be detected by the kit in cases with a sufficient viral load. On the other hand, the genotypes of 20 samples, which were ELISA positive and seminested PCR positive, were 7 GII/3 and 13 GII/4. It would appear that these samples have a smaller viral load than monoplex PCR-positive stools. This suggests that there are other factors, such as inhibitors, that may cause the lower sensitivity of ELISA.
The sensitivity, specificity, and agreement of the kit were superior to those of the Denka and Dako kits (1). Dimitriadis and Marshall showed in 2005 that the RIDASCREEN ELISA kit could not be recommended for the study of stool samples in Australian outbreaks (2). In that report, the sensitivity and specificity of the kit were 71 and 47%, respectively, with the same cutoff calculations as our own. The difference between their sensitivity value and our own, which was 76.3%, was not large. On the other hand, the specificity was very different. In the present study, the specificity of the kit based on RT-PCR assay was 94.9%. There were the false-positive samples in their results. The reason for the difference in the specificities is unclear. We have been unable to obtain either the Denka kit or the Dako kit and have not been able to compare the RIDASCREEN kit with these kits using the same stool samples.
In conclusion, our results indicated that the kit could be a useful tool for sporadic diarrheal samples. However, it is quite possible to contain many kinds of genotypes in diarrheal samples derived from food-borne sources, and the particular kinds of genotypes found in such cases are not always the same as the genotypes found in sporadic cases. All in all, the reactivity for GII/3 and GII/6 needs to be improved in order to facilitate the detection of etiological agents in outbreaks.
ACKNOWLEDGMENTS
This study was supported by Grants-in-Aid from the Ministry of Education and Sciences of Japan.
We thank T. Kaneshi, A. Yamamoto, S. Nishimura, S. Nakaya, and T. Nishimura for collecting stool samples.
FOOTNOTES
Corresponding author. Mailing address: Department of Developmental Medical Sciences, School of International Health, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan. Phone: 81-3-5841-3590. Fax: 81-3-5841-3629. E-mail: mshoko@mail.ecc.u-tokyo.ac.jp.
REFERENCES
Burton-MacLeod, A., E. M. Kane, R. S. Beard, L. A. Hadley, R. I. Glass, and T. Ando. 2004. Evaluation and comparison of two commercial enzyme-linked immunosorbent assay kits for detection of antigenically diverse human noroviruses in stool samples. J. Clin. Microbiol. 42:2587-2595.
Dimitriadis, A., and J. A. Marshall. 2005. Evaluation of a commercial enzyme immunoassay for detection of norovirus in outbreak specimens. Eur. J. Clin. Microbiol. Infect. Dis. 24:615-618.
Dimitriadis, A., L. D. Bruggink, and J. A. Marshall. 2006. Evaluation of the Dako IDEIA norovirus EIA assay for detection of norovirus using faecal specimens from Australian gastroenteritis outbreaks. Pathology 38:157-165.
Reference deleted.
Hansman, G. S., L. T. P. Doan, T. A. Kguyen, S. Okitsu, K. Katayama, S. Ogawa, K. Natori, N. Takeda, Y. Kato, O. Nishio, M. Noda, and H. Ushijima. 2004. Detection of norovirus and sapovirus infection among children with gastroenteritis in Ho Chi Minh City, Vietnam. Arch. Virol. 149:1673-1688.
Kageyama, T., M. Shinohara, K. Uchida, S. Fukushi, F. B. Hoshino, S. Kojima, R. Takai, T. Oka, N. Takeda, and K. Katayama. 2004. Coexistence of multiple genotypes, including newly identified genotypes, in outbreaks of gastroenteritis due to norovirus in Japan. J. Clin. Microbiol. 42:2988-2995.
Kirkwood, C. 2004. Viral gastroenteritis in Europe: a new norovirus variant Lancet 363:671-672.
Kirkwood, C. D., R. Clark, N. Bogdanovic-Sakran, and R. F. Bishop. 2005. A 5-year study of the prevalence and genetic diversity of human caliciviruses associated with sporadic cases of acute gastroenteritis in young children admitted to hospital in Melbourne, Australia (1998-2002). J. Med. Virol. 77:96-101.
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Maunula, L., and C.-H. Von Bonsdorff. 2005. Norovirus genotypes causing gastroenteritis outbreaks in Finland 1998-2002. J. Clin. Virol. 34:186-194.
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Phan, T. G., T. Kuroiwa, K. Kaneshi, Y. Ueda, S. Nakaya, S. Nishimura, A. Yamamoto, K. Sugita, T. Nishimura, F. Yagyu, S. Okitsu, W. E. G. Muller, N. Maneekarn, and H. Ushijima. 2006. Changing distribution of norovirus genotypes and genetic analysis of recombinant GIIb among infants and children with diarrhea in Japan. J. Med. Virol. 78:971-978.
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Richards, A. F., B. Lopman, A. Gunn, A. Curry, D. Ellis, H. Cotterill, S. Ratcliffe, M. Jenkins, M. Appleton, C. I. Gallimore, J. J. Gray, and D. W. G. Brown. 2003. Evaluation of a commercial ELISA for detection Norwalk-like virus antigen in faeces. J. Clin. Virol. 26:109-115.
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Nippon Becton Dickinson Company, Tokyo, Japan
Institüt fur Physiologische Chemie, Abteilung Angewandte Molekularbilogie, Johannes Gutenberg-Universitat, Mainz, Germany
ABSTRACT
The commercial norovirus enzyme-linked immunosorbent assay kit was evaluated for its reactivity to recombinant virus-like particles and the detection of natural viruses from stool samples of Japanese infants and children with sporadic acute gastroenteritis compared to reverse transcription-PCR. The kit had a sensitivity of 76.3% and a specificity of 94.9%. Our results clearly indicated that the kit allows the detection of the most prevalent genotype, GII/4. In order to increase the sensitivity of the kit, the reactivity with norovirus of GII/3 and GII/6 genotypes needs to be improved.
TEXT
Norovirus (NoV) is one of the leading etiologic agents of nonbacterial sporadic acute gastroenteritis (AGE) in infants and children, and outbreaks of this infection may be due to contaminated water or food. At present, the reverse transcription-PCR (RT-PCR) assay is widely used to detect NoV in diarrheal stool samples. The development of immunological methods to detect NoV has been delayed due to the lack of viruses in cell culture and to diverse genotypes with distinct antigenicities. NoVs are currently divided into five genogroups, and most human NoV strains belong to two genogroups: genogroup I (GI) and genogroup II (GII). Furthermore, each genogroup contains at least 15 and 18 genotypes, respectively (13). RT-PCR is an expensive and complicated technique, and its use requires special equipment and skills. Thus, a faster and simpler method is needed. At present, three commercial enzyme-linked immunosorbent assay (ELISA) kits are available, the IDEIA NLV kit from Dako Cytomation, Ltd. (Ely, United Kingdom), the SRSV(II)-AD kit from Denka Seiken Co., Ltd. (Tokyo, Japan), and the RIDASCREEN norovirus (R-Biopharm AG, Darmstadt, Germany). According to previous evaluations of the ELISA kits, the first two kits cannot effectively replace RT-PCR for NoV detection due to their low sensitivities and/or specificities (1, 3, 19). To date, the RIDASCREEN norovirus assay has only been evaluated by one Australian group using outbreak specimens (2). No research has hitherto been conducted using recombinant virus-like particles (rVLPs) and sporadic stool samples.
Therefore, using the RIDASCREEN norovirus ELISA kit, we set out to measure the reactivity of 16 kinds of rVLPs, to detect the presence of NoV in fecal samples from infants and children with sporadic AGE in Japan, and to compare the sensitivity and specificity data with those obtained with the RT-PCR.
We previously expressed one rVLP (strain 1207, GII/4) (14). The other 15 rVLPs were prepared from NoV isolated from stool samples among infants and children with diarrhea between 1995 and 2003 in Japan. The genotypic classification of these NoV was performed based on the method described by Kageyama et al. (6). These are genotypes 1 (strain 4656), 3 (strain 3634), 4 (strain 2876), 8 (strain 3006), and 11 (strain 2258) in genogroup I and genotypes 1 (strain 3101), 2 (strain 2840), 3 (strain 3229), 5 (strain 3611), 6 (strain 3612), 7 (strain 419), 12 (strain 2087), 13 (strain 3385), 14 (strain 2468), and 15 (strain 3625) in genogroup II. The production of recombinant bacmids was performed using the baculovirus expression system with Gateway Technology (Invitrogen Japan, Tokyo) and the transfection of bacmids into insect cells, as well as the purification of rVLPs, was performed according to the method of Hansman et al. (5). We used two sense primers: attB1NVGI (GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT CGA AGG AGA TAG AAC CAT GAT GAT GGC GTC TAA GG) for GI strains and attB1NVGII (GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT CGA AGG AGA TAG AAC CAT GAA GAT GGC GTC GAA TGA) for GII strains. Purified rVLPs from the cultured supernatants of the inset cells were examined for particle formation by electron microscopy. Protein concentration of each rVLP was measured by BCA Coomassie protein assay (Pierce Biotechnology, Inc., Rockford, IL), and 150 μg/ml was prepared as stock solutions. The assays were started from 10 μg/ml as the highest concentration.
The rVLPs stock solutions were serially threefold diluted with the sample dilution buffer in the the RIDASCREEN norovirus ELISA kit and used to determine the minimal concentration of each rVLP for detection by ELISA according to the manufacturer's manual. All of the assays except that for GI/3 were done with kits of the same lot number. In the manual, the cutoff value is calculated as an absorbance value of negative control plus 0.15. Values that are 10% above or below the cutoff value are considered to be in the gray zone and therefore need to be examined again. In view of this, the theoretical minimal detectable concentration of each rVLP was determined as a calculated value which gave an absorbance value that was 10% above the cutoff value in each assay. Each assay was conducted in triplicate, and the experiment for each rVLP was repeated three to six times.
Five hundred and three stool samples were collected from infants and children with AGE who visited six pediatric clinics in Sapporo, Tokyo, Maizuru, and Osaka, Japan, from July 2004 to March 2005. All of the stool samples were stored at –30°C until testing. Watery stool samples were diluted 1:2 with phosphate-buffered saline (PBS), and hard stool samples were suspended to 1:5 with PBS. The suspensions were clarified by centrifugation at 10,000 x g for 15 min. The supernatants were diluted to 1:3 with the sample dilution buffer of the kit and used for the assay. The positives or negatives of the samples were determined as mentioned above.
Ten percent stool suspensions of 503 samples were prepared with PBS from the same aliquots for ELISA, and viral RNA was extracted by the QIA amp viral RNA mini kit (QIAGEN, Tokyo, Japan). The detection of NoV (GI and GII), astrovirus, sapovirus, rotavirus, and adenovirus was performed by two sets of multiplex PCR (21, 22). NoV-negative samples were examined by using two sets of monoplex PCRs, for NoV GI and GII. Twenty samples were further assayed by seminested PCR using a primer set, which were G2SKF and G2SKR for NoV GII (9). The genotypes of NoV were determined according to the method of Phan et al. (16).
The minimal detectable sensitivity is indicated in Table 1. The kit could detect GI/1 and GII/4 rVLPs at concentrations of <0.01 μg/ml. rVLPs of GI/3, GI/8, GI/11, GII/1, GII/2, and GII/12 were detectable within a range between 0.04 and 1 μg/ml. On the other hand, rVLPs of GI/4, GII/3, GII/5, GII/6, GII/14, and GII/15 were detected at more than 1 μg/ml. In cases where the assays for GI/4, GII/3, GII/6, GII/14, and GII/15 could not be detected at concentrations of <10 μg/ml and theoretical detectable concentrations could not be calculated, minimal concentrations given by the assay have been indicated (Table 1). GII/6 rVLP could be detected once at the highest concentration, 10 μg/ml. Two rVLPs of GII/7 and GII/13 could not be detected at a concentration of <10 μg/ml.
NoV in stool samples collected from sporadic cases in Japan was examined using both the ELISA kit and the RT-PCR, and the kit was evaluated based on the RT-PCR (Table 2). The calculated percent sensitivity, specificity, and agreement were 76.3, 94.9, and 90.7%, respectively. Twenty samples were determined to be positive by the kit but negative by the RT-PCR. These samples became positive when tested by the seminested PCR using NoV GII-specific primer pair. A total of 27 samples were positive with the RT-PCR but negative with the kit. The genotypes of 134 positive stool samples recorded by the RT-PCR were identified by using the clustering determined by Kageyama et al. (6) (Table 3). The genotypes of kit-positive, PCR-positive samples were 1 GI/1, 3 GII/3, 82 GII/4, and 1 GII/6, and the sensitivities of GI/1, GII/3, GII/4, and GII/6 were 50, 23.1, 85.4, and 33.3%, respectively. The low sensitivities of GII/3 and GII/6 were comparable to the results for the rVLPs. RT-multiplex PCR detected four other species of viruses in 503 stool samples. These were 7 group A rotavirus, 27 adenovirus, 30 sapovirus, and 1 astrovirus samples, and the stool samples containing these viruses were determined to be negative by ELISA. Furthermore, multiplex-PCR indicated that 8 of 112 NoV GII-positive samples were mixed infected with other viruses (5 sapovirus, 2 group A rotavirus, and 1 adenovirus).
Some studies showed that the strains belonging to GII/4 cluster were most predominant not only in stool samples from sporadic cases involving infants and children but also from the outbreaks (8, 10, 12, 15, 16, 19). On the other hand, it was found that various genotypes of NoV strains were detected in the outbreak cases, and there were no predominant genotypes in outbreak strains (20). Furthermore, a change in the distribution of NoV genotypes in the sporadic cases and the emergence of recombinant viruses has been reported (7, 11, 17, 18).
The ELISA kit could detect two kinds of rVLPs (GI/1 and GII/4) with a high sensitivity. Meanwhile, the GII/3 and GII/6 rVLPs formed a group that was responsive at higher concentrations. A total of 23.1% of the stool samples containing GII/3 NoV, and 33.3% of the samples with the GII/6 genotype could be effectively examined by the kit. NoV genotypes with low reactivity levels in the stool samples could be detected by the kit in cases with a sufficient viral load. On the other hand, the genotypes of 20 samples, which were ELISA positive and seminested PCR positive, were 7 GII/3 and 13 GII/4. It would appear that these samples have a smaller viral load than monoplex PCR-positive stools. This suggests that there are other factors, such as inhibitors, that may cause the lower sensitivity of ELISA.
The sensitivity, specificity, and agreement of the kit were superior to those of the Denka and Dako kits (1). Dimitriadis and Marshall showed in 2005 that the RIDASCREEN ELISA kit could not be recommended for the study of stool samples in Australian outbreaks (2). In that report, the sensitivity and specificity of the kit were 71 and 47%, respectively, with the same cutoff calculations as our own. The difference between their sensitivity value and our own, which was 76.3%, was not large. On the other hand, the specificity was very different. In the present study, the specificity of the kit based on RT-PCR assay was 94.9%. There were the false-positive samples in their results. The reason for the difference in the specificities is unclear. We have been unable to obtain either the Denka kit or the Dako kit and have not been able to compare the RIDASCREEN kit with these kits using the same stool samples.
In conclusion, our results indicated that the kit could be a useful tool for sporadic diarrheal samples. However, it is quite possible to contain many kinds of genotypes in diarrheal samples derived from food-borne sources, and the particular kinds of genotypes found in such cases are not always the same as the genotypes found in sporadic cases. All in all, the reactivity for GII/3 and GII/6 needs to be improved in order to facilitate the detection of etiological agents in outbreaks.
ACKNOWLEDGMENTS
This study was supported by Grants-in-Aid from the Ministry of Education and Sciences of Japan.
We thank T. Kaneshi, A. Yamamoto, S. Nishimura, S. Nakaya, and T. Nishimura for collecting stool samples.
FOOTNOTES
Corresponding author. Mailing address: Department of Developmental Medical Sciences, School of International Health, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan. Phone: 81-3-5841-3590. Fax: 81-3-5841-3629. E-mail: mshoko@mail.ecc.u-tokyo.ac.jp.
REFERENCES
Burton-MacLeod, A., E. M. Kane, R. S. Beard, L. A. Hadley, R. I. Glass, and T. Ando. 2004. Evaluation and comparison of two commercial enzyme-linked immunosorbent assay kits for detection of antigenically diverse human noroviruses in stool samples. J. Clin. Microbiol. 42:2587-2595.
Dimitriadis, A., and J. A. Marshall. 2005. Evaluation of a commercial enzyme immunoassay for detection of norovirus in outbreak specimens. Eur. J. Clin. Microbiol. Infect. Dis. 24:615-618.
Dimitriadis, A., L. D. Bruggink, and J. A. Marshall. 2006. Evaluation of the Dako IDEIA norovirus EIA assay for detection of norovirus using faecal specimens from Australian gastroenteritis outbreaks. Pathology 38:157-165.
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