Simultaneous Detection of Seven Enteric Viruses Associated with ...

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Feb 8, 2012 - Yan Liu, Zi-qian Xu, Qing Zhang, Miao Jin, Jie-mei Yu, Jin-song Li, Na Liu, Shu-xian Cui, Xiang-yu Kong, Hong Wang, Hui-ying Li,. Wei-xia ...
Simultaneous Detection of Seven Enteric Viruses Associated with Acute Gastroenteritis by a Multiplexed Luminex-Based Assay Yan Liu, Zi-qian Xu, Qing Zhang, Miao Jin, Jie-mei Yu, Jin-song Li, Na Liu, Shu-xian Cui, Xiang-yu Kong, Hong Wang, Hui-ying Li, Wei-xia Cheng, Xue-jun Ma, and Zhao-jun Duan State Key Laboratory for Molecular Virology and Genetic Engineering, National Institute for Viral Disease Control and Prevention, China CDC, Beijing, China

Rapid and broad diagnostic methods are needed for the identification of viral agents of gastroenteritis. In this study, we used Luminex xMAP technology to develop a multiplexed assay for the simultaneous identification of major enteric viral pathogens, including rotavirus A (RVA), noroviruses (NoVs) (including genogroups GI and GII), sapoviruses (SaV), human astrovirus (HAstV), enteric adenoviruses (EAds), and human bocavirus 2 (HBoV2). The analytical sensitivity allowed detection of 103 (EAds, HBoV2, and RVA) and 104 (NoV GI and GII, SaV, and HAstV) copies per reaction mixture. Compared to conventional PCR, the Luminex-based assay yielded greater than 75% sensitivity and 97% specificity for each virus, and the kappa correlation for detection of all viruses ranged from 0.75 to 1.00. In conclusion, this multiplexed Luminex-based assay provides a potentially rapid, high-throughput, and maneuverable diagnostic tool for major viral pathogens associated with gastroenteritis.

V

iral gastroenteritis is a major health problem and continues to be a crucial cause of morbidity and mortality in developing countries (4, 12). In developed countries, mortality is low, but morbidity and economic consequences due to viral gastroenteritis are significant (23). The most common viral pathogens known to cause human acute gastroenteritis are rotavirus A (RVA), human caliciviruses, enteric adenoviruses (EAds), and human astrovirus (HAstV) (30). Recent studies have suggested that other enteric viruses, such as human parechovirus (HPeV), human picobirnavirus (HPBV), human bocavirus (HBoV), and Aichi virus, are also associated with acute gastroenteritis (6, 13, 15). Methods for accurate diagnosis and tracking of viral pathogens are required to develop appropriate management strategies to mitigate morbidity and mortality due to gastroenteritis. Conventional and real-time reverse transcription-PCR (RTPCR) protocols for detection of these viruses have been published, but few of them allow for simultaneous detection of all major enteric viruses in one assay. Luminex xMAP technology offers a novel platform for high-throughput nucleic acid detection and is being used in a variety of applications (2, 10, 20, 26, 32). In this study, we used xMAP technology to develop a multiplexed assay for simultaneous identification of major enteric viral pathogens. The analytical and clinical sensitivities and specificities of this assay were evaluated with 140 fecal samples from infants with diarrhea and compared to those of conventional PCR. MATERIALS AND METHODS Sample collection. Two panels of samples were used in this study. The first panel consisted of 10 samples in which RVA, norovirus (NoV) genogroups GI and GII, sapovirus (SaV), HAstV, EAds, HBoV2, enterovirus 71 (EV71), HPeV, and HPBV II were previously identified by PCR with sequencing and/or enzyme-linked immunosorbent assay (ELISA) (5, 9, 21, 25). This panel was used for initial Luminex assay development and optimization. The second panel of samples consisted of 140 clinical fecal specimens collected from children hospitalized with diarrhea at the First Hospital of Lanzhou University between July 2008 and June 2009. These samples were used to further evaluate the assay. DNA and RNA extraction. Viral DNA and RNA were extracted from 450 ␮l of 10% fecal suspension in phosphate-buffered saline by using the

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Viral Nucleic Acid Extraction Kit II (Geneaid Biotech Ltd., Taipei, Taiwan) according to the manufacturer’s instructions. The elution volume was 50 ␮l. Extracted DNA or RNA was subdivided into 5-␮l volumes and stored at ⫺80°C. Viral reference preparation. We chose a conservative gene of each virus as a target segment for the viral reference. The PCR fragments from samples positive for RVA (VP6), NoV GI (RNA-dependent RNA polymerase [RDRP]) and GII (RDRP), SaV (polymerase), HAstV (ORF1a), EAds (hexon), and HBoV2 (NP1) were purified using a QIAquick PCR purification kit (Qiagen) and were identified by sequencing. Then, the cDNA was ligated into a pGEM-T Easy Vector system (Promega) and cloned in DH5␣ Chemically Competent Cell (TransGen Biotech, Beijing). Plasmids of EAds and HBoV2 were purified and sequenced. Plasmids of RVA, NoV GI and GII, SaV, and HAstV were purified, sequenced, linearized with PvuII (New England Biolabs), and purified again. Products were transcribed into RNA with the T7 RiboMAX Express RNAi system or the RiboMAX large scale RNA production systems SP6/T7 kit (Promega Biosciences, Inc.). After DNase I digestion, RNA transcripts were purified with a QIAquick PCR purification kit (Qiagen) and quantified in a spectrophotometer according to the following formulas: number of copies (DNA) ⫽ (amount · 6.022 ⫻ 1023)/(length · 1 ⫻ 109 · 650), and number of copies (RNA) ⫽ (amount · 6.022 ⫻ 1023)/(length · 1 ⫻ 109 · 345); then, serial 10-fold dilutions were performed. The in vitro-transcribed RNA and plasmid DNA were then used as quantification standards to determine the sensitivity of the multiplexed Luminex-based assay. Diethylpyrocarbonate (DEPC) water was used as a negative control. Design of multiplex PCR primers and specific probes. Gene-specific primers and specific capture probes were designed based on sequence information obtained from the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/) and using Lasergene 8.0 (DNASTAR Inc., Madison, WI) and PrimerPlex 2 software (Premier Biosoft, Palo Alto, CA). For target-enriched multiplexing PCR (Tem-PCR),

Received 29 December 2011 Returned for modification 8 February 2012 Accepted 10 April 2012 Published ahead of print 18 April 2012 Address correspondence to Zhao-jun Duan, [email protected], or Xue-jun Ma, [email protected]. Copyright © 2012, American Society for Microbiology. All Rights Reserved. doi:10.1128/JCM.06790-11

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Forward

GTACGACTCACTATAGGGAARRTTICCAATTCCTCCAGT GTACGACTCACTATAGGGAGCAAGAGGGTCAGAAGCATT GTACGACTCACTATAGGGATTTCTAATCCAGGGGTCAAT GTACGACTCACTATAGGGAACYTCAAAVSTACCBCCCCA GTACGACTCACTATAGGGACATGTGCTGCTGTTACTATG GTACGACTCACTATAGGGATTATAGGCGGTTCCGGAGTA GTACGACTCACTATAGGGAAGTGCCATCTCTAGCAAGCC GTACGACTCACTATAGGGA

Reverse

TABLE 1 Primers and probes designed for the multiplexed Luminex-based assay developed in this study

Target virus (protein)

AGGTGACACTATAGAATAAAGTCTTCRACATGGAKGT AGGTGACACTATAGAATACGCTGGATGCGCTTCCATGA AGGTGACACTATAGAATACAGACAAGAGCCAATGTTCA AGGTGACACTATAGAATACAATCCAATCCAATGTCCCT AGGTGACACTATAGAATACGTCATTATTTGTTGTCATA AGGTGACACTATAGAATAAGACAGGTCACAGCGACTGA AGGTGACACTATAGAATATGCTTCGAAGACCTCAGACC AGGTGACACTATAGAATA

Primer sequence (5=¡3=)a

RVA (VP6) NoV GI (RDRPb) NoV GII (RDRP) SaV (polymerase) HAstV (ORF1a) EAds (hexon) HBoV2 (NP1) Universal primer 181 162 279 220 326 231 205

Size (bp)

ATAGTAACCATGAACGGAAA TTAATACAGCTGACCCCTTA TCCCAGTTTTGTGAATGAAG ATTAACCCGTACACTTCTCA TGATGCTAATGGGAAGGTTG GCACGAATCGCAGCGTCAGT AGTCAACACAGGGCTAATCA

Probe sequence

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Degenerate primer abbreviations are as follows: B, G/C; R, A/G; S, C/G; Y, C/T; V, A/C/G. Underlined oligonucleotides are universal sequences. RDRP, RNA-dependent RNA polymerase.

Specificity of the Luminex-based multiplexed assay. The specificity of our Luminex-based multiplexed assay was examined by testing, in triplicate, 10 different pathogens, including seven target viruses and three other viruses (EV71, HPeV, and HPBV II). TE buffer was used as a blank control, and DEPC water was used as a negative control. As shown in Fig. 1, the assay was able to detect all target enteric viruses, whereas no signal corresponding to EV71, HPeV, HPBV II, the blank control, or negative control was detected. To further test the performance of this assay, the

a

RESULTS

b

sets of gene-specific primers were selected to amplify 100 to 300 bp targeting conservative gene regions using a melting temperature range of 55 to 60°C, a length range of 18 to 22 bp, and a GC content of 30 to 70%. The primers included tag sequences that can be recognized by universal primers called SuperPrimers (Qiagen, Valencia, CA). The reverse SuperPrimer was labeled with biotin-TEG at its 5= end. For microsphere-based Luminex detection, specific capture probes were designed to identify 20-nucleotide sequences at a melting temperature range of 55 to 60°C. Each probe was synthesized with 5= Amino Modifier C12 to enable coupling to the carboxyl group located on the microsphere. For all primer and probe sequences, BLAST analysis using the nucleotide database (NCBI) was performed to ensure specificity. All primers and probes were purified by high-pressure liquid chromatography (HPLC) (Table 1). Tem-PCR amplification. After confirming that all of the individual primer pairs allowed for amplification of all seven target viruses in a single PCR, forward and reverse gene-specific primers were mixed together. RNA and DNA were put together for the reaction from the first step of the multiplex PCR. After the Tem PCR amplification conditions were optimized, the multiplex PCR mixtures included 3 ␮l RNA or DNA template mixed with 4 ␮l 5⫻ RT-PCR buffer, 0.8 ␮l enzyme mix (OneStep RT-PCR kit; Qiagen), 400 ␮M deoxynucleoside triphosphates (dNTPs), 1.5 ␮l reverse primer mix (50 to 160 nM), 1.5 ␮l forward primer mix (50 to 160 nM), 0.8 ␮l forward SuperPrimer (10 ␮M), 1.0 ␮l biotin-labeled reverse SuperPrimer (10 ␮M), 5 to 10 U RNase inhibitor, and RNase-free water in a final volume of 20 ␮l. Amplification was carried out using a six-stage cycling program: (i) reverse transcription at 50°C for 30 min; (ii) hot start at 95°C for 15 min; (iii) enrichment stage of 15 cycles at 94°C for 45 s, 50°C for 1 min, and 72°C for 1 min; (iv) tagging stage of 5 cycles at 94°C for 15 s and 65°C for 15 s; (v) amplification stage of 20 cycles at 94°C for 30 s, 46°C for 30 s, and 72°C for 30 s; and (vi) a final extension at 72°C for 3 min. Hybridization and Luminex analysis. The specific capture probes were bound to different polystyrene microspheres following the Bio-Plex Bead Coupling Protocol (7, 19, 27, 34). Biotinylated PCR products were hybridized to a fluid microsphere-based array in wells of a 96-well plate and detected using streptavidin-phycoerythrin. To optimize the hybridization conditions, five different hybridization temperatures (46°C, 48°C, 50°C, 52°C, and 55°C) and incubation times (15, 30, 45, 60, and 90 min) were tested. The optimal hybridization temperature and incubation time proved to be 50°C and 30 min, respectively. The working microsphere mixture consisted of seven types of beads, each coupled to a unique gene-specific capture probe. A total of 5 ␮l biotin-labeled PCR product was directly mixed with 33 ␮l working microsphere mixture and 12 ␮l Tris-EDTA (TE) buffer (pH 8.0). Hybridization was carried out at 95°C for 3 min followed by 50°C for 30 min. The hybridization product was then transferred to a preheated filter plate and quickly removed by applying a vacuum pressure of 25 to 50 mm Hg. For detection, 100 ␮l streptavidin–R-phycoerythrin (3 ␮g/ml) in 1⫻ tetramethylammonium chloride (TMAC) was added to each well, followed by incubation for 5 min at 50°C. Finally, the signals produced for each bead were analyzed using the Luminex 200 (Luminex Corporation, Austin, TX) and expressed as mean fluorescence intensity (MFI). The cutoff value for a positive result was set at three times the mean MFI value of the negative control.

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FIG 1 Analytic specificity of the multiplexed Luminex assay. The analysis of the specificity of the multiplexed Luminex assay was carried out with EV71, HPeV, PBV, rotavirus A, NoV GI, NoV GII, SaV, HAstV, EAds, and HBoV2. TE buffer was used as a blank control, and DEPC water was used as a negative control. Biotin-labeled PCR products were separated by probe-coupled beads and are presented in terms of dye signal median fluorescence intensity (MFI) in arbitrary units on the y axis. Each peak was identified by beads coupled with specific capture probes and is indicated on the z axis.

presence of multiple targets was tested in combination (Fig. 2). The ability to detect infections with multiple pathogens is important because such infections occur naturally and are often difficult to detect. Detection sensitivity of the multiplexed Luminex-based assay. The limit of detection for each of the target viruses was determined by testing triplicates of six serial 10-fold dilutions of each reference virus. The limiting dilution was defined as that containing the fewest virus genomic copies that still gave a positive result for all replicates. As shown in Table 2, the limiting dilution for EAds and HBoV2 was 103 copies per reaction and that for RVA,

NoV GI and GII, SaV, and HAstV was 104 copies per reaction mixture. Intra- and interassay reproducibilities of the multiplexed Luminex-based assay. To confirm the reproducibility of this method, intra-assay (each sample tested three times within an experiment) and interassay (each sample tested one time in three different experiments) precision was evaluated (28). For each reference virus, 105-copy dilutions were mixed. These dilutions were used multiple times to confirm the consistency of the viral concentration every time. The coefficient of variation (CV) for each target virus within an experiment ranged from 0.11% to 1.44%, and that between experiments ranged from 1.00% to 4.17% (Table 3). Evaluation of clinical specimens. To evaluate the assay’s performance using clinical specimens, we tested 140 fecal samples using the Luminex-based multiplexed assay and conventional RTPCR (RT-PCR for RVA, NoV, SaV, and HAstV, and PCR for EAds and HBoV2) in parallel (Table 4). NoV GI was not detected by either method. Two RVA samples had discrepant results between the Luminex and PCR assays. Four NoV GII samples, one SaV sample, two HAstV samples, and five EAd samples were positive

TABLE 2 Analytic sensitivity of the multiplexed Luminex-based assay

FIG 2 Multiple-target detection by the Luminex-based multiplexed assay. The analysis of the mixed-sample detection capacity of the Luminex-based multiplex assay was carried out using four sets of random mixed samples. Mix 1 included HAstV, rotavirus A, and EAds. Mix 2 included NoV GI, NoV GII, and sapovirus. Mix 3 included rotavirus A, NoV GII, and HBoV2. Mix 4 included HAstV, EAds, and HBoV2. DEPC water was used as a negative control. Biotin-labeled PCR products were separated by probe-coupled beads and are presented in terms of dye-signal average median fluorescence intensity (MFI) in arbitrary units on the y axis. Error bars represent 5% of the MFI value.

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Target virus RVA NoV GI NoV GII SaV HAstV EAds HBoV2

No. of positive samples/no. of samples tested with the following no. of virus genomic copies per reaction mixture: 10

102

103

104

105

106

Analytical sensitivity (no. of copies/reaction mixture)

0/3 0/3 0/3 0/3 0/3 0/3 0/3

0/3 0/3 0/3 0/3 0/3 0/3 0/3

0/3 0/3 2/3 0/3 1/3 3/3 3/3

3/3 3/3 3/3 3/3 3/3 3/3 3/3

3/3 3/3 3/3 3/3 3/3 3/3 3/3

3/3 3/3 3/3 3/3 3/3 3/3 3/3

104 104 104 104 104 103 103

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TABLE 3 Intra- and interassay reproducibilities of the multiplexed Luminex-based assay Intra-assay reproducibility

Interassay reproducibility

MFI with the following no. of replicates

MFI with the following no. of experiments

Virus

1

2

3

CV (%)

1

2

3

CV (%)

RVA NoV GI NoV GII SaV HAstV EAds HboV2

4,162.5 2,912.0 2,256.5 2,948.5 3,280.5 3,008.0 4,238.0

4,210.5 3,004.0 2,251.0 2,953.0 3,219.0 3,012.5 4,211.5

4,194.5 3,001.5 2,251.5 2,938.5 3,201.5 3,016.5 4,181.5

0.48 1.44 0.11 0.21 1.05 0.12 0.55

4,549.0 3,012.5 1,956.5 2,648.5 3,383.5 3,558.0 5,238.0

4,692.5 3,214.0 2,051.0 2,453.0 3,119.0 3,652.5 5,111.5

4,794.5 3,114.5 1,851.5 2,538.5 3,233.5 3,662.0 5,181.0

2.15 2.64 4.17 3.14 3.34 1.30 1.00

by PCR but negative by the multiplexed Luminex-based assay. Two HAsV samples and three EAd samples identified as negative by PCR tested positive by the multiplexed Luminex-based array. In addition, five samples identified as coinfections of RVA and HAstV, three of RVA and EAds, three of RVA and NoV GII, and one of RVA, HAstV, and NoV GII were detected by both methods. Based on the 140 clinical samples tested, the sensitivities (true positives) of detection of the different viruses were 100% (RVA), 86.67% (NoV GII), 80% (SaV), 88.24% (HAstV), 76.19% (EAds), and 100% (HBoV2), and the specificities (true negatives) were 97.98%, 100%, 100%, 98.37%, 97.48%, and 100%, respectively. Concordance between the Luminex-based multiplexed assay and

conventional PCR results for all viruses was ⬎90%, and the kappa correlation was ⬎0.75. DISCUSSION

Viral gastroenteritis is responsible for a significant proportion of the worldwide diarrheal disease burden. RVA is the principal cause of severe diarrhea, with approximately 95% of children experiencing rotavirus gastroenteritis by 5 years of age (1, 36). Noroviruses (NoVs) and sapoviruses (SaVs) are members of the family Caliciviridae. NoVs are now recognized as the leading cause of epidemic viral gastroenteritis, and the genogroups GI, GII, and occasionally GIV are associated with human infection (17, 22, 24).

TABLE 4 Performance of the multiplexed Luminex-based assay compared to RT-PCR/PCRa Virus and multiplexed Luminex-based assay result

No. of samples RT-PCR/PCR: Positive

Negative

RVA Positive Negative

41 0

2 97

NoV GI Positive Negative

0 0

0 140

NoV GII Positive Negative

26 4

0 110

SaV Positive Negative

4 1

0 135

HAstV Positive Negative

15 2

2 121

EAds Positive Negative

16 5

3 116

HBoV2 Positive Negative

2 0

0 138

Sensitivity (%)

Specificity (%)

Agreement (%)

Kappa value

100

97.98

98.57

0.97

86.67

100

97.14

0.91

80

100

99.29

0.89

88.24

98.37

97.14

0.87

76.19

97.48

94.29

0.77

100

100

100

1.00

a The numbers of positive and negative samples detected by either testing method are shown. Sensitivity and specificity indicate true positive and true negative results, as determined by conventional PCR.

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HAstV and EAds are also recognized as important viral agents of diarrhea in infants and the elderly (31). Although the association between HBoV2 and gastroenteritis is still unknown, the virus has been frequently found in fecal samples from children with gastroenteritis (3, 8, 18). The accurate diagnosis of enteric viruses is important for clinical care of viral gastroenteritis. Given that many clinical laboratories do not routinely test for the panel of viruses discussed in this article, it is worth a few more sentences to expand on how clinical care will change based on this new information and whether the changes in clinical care will be different in different countries. When dealing with public health crises, accurate diagnoses are important for epidemiologic research as well as clinical care of patients. Previous studies have found at least one viral agent in nearly 43% of clinical diarrheal specimens, with multiple agents detected in 11% (29). Over the past several years, various detection methods, including cell culture, neutralization tests, antigen detection by immunofluorescence, and PCR, have been widely used for virus identification. Recently, multiplex analysis has become more and more popular. However, multiplexed molecular assays are subject to concerns of decreased sensitivity and specificity related to nonspecific or incompatible amplification, incompatible primer sets, high background, and poor reproducibility (21, 33). In addition, coinfections are hard to detect, as one virus, usually one at a high titer, often dominates detection assays, especially in cases of single-pass comparative immunological methods. Target-enriched multiplexing PCR (Tem-PCR) addresses these issues by its novel amplification strategy and the application of Luminex xMAP technology (11, 14, 16, 35). To improve the diagnostic methods for multiplex detection of viral pathogens in diarrhea, we established a novel multiplex PCR technology that allows accurate and rapid detection of multiple pathogens in one sample and therefore provides a molecular differential diagnosis. By using such technology, a public health laboratory can quickly rule out (or rule in) an array of possible pathogens at the initial stage of disease outbreak. In comparing results of the Luminex-based multiplex assay and conventional PCR, we found that there was no significant difference between the detection rates (measurement agreement was ⬎90% for all of the tested viruses) in the clinical evaluation. The sensitivity for each virus was ⬎76%, and the specificity was ⬎96%. Statistical analysis found that the kappa correlation between the two methods was ⬎0.75. This indicates that the Luminex-based multiplex assay is almost as specific as the conventional PCR assay. Moreover, it can accurately and rapidly detect multiple pathogens in one sample, which could potentially correlate with clinical significance. Antibiotic abuse is severe in the clinical care of viral gastroenteritis, and the accurate diagnosis of enteric viruses is important for clinical care of gastroenteritis. Our multiplexed Luminex-based assay has the ability to detect coinfections with a single test and can be performed in less than 6 h, from sample preparation to obtaining the results. Another advantage of this assay is that one tube allows for the identification of seven enteric viruses associated with gastroenteritis. Thus, the potential for cross contamination is, theoretically, much lower, as is the cost of specimen testing, making it more useful in most clinical settings. Our assay exhibited high sensitivity, specificity, and reproducibility with both analytical and clinical specimens. It should be noted that there are not sufficient samples positive for

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SaV and for HBoV2 to determine meaningful statistics in our study. With the limited specimens available at this time, only preliminary findings were reported in this study. Future priorities should include more specimens to evaluate this novel method. Our results indicate that our Luminex-based multiplexed assay has potential as a rapid and cost-effective diagnostic tool for viral gastroenteritis. Furthermore, this assay can be expanded to include more specific probes to identify other viral or bacterial agents of gastroenteritis or other diseases. ACKNOWLEDGMENTS This work was supported by the China Mega-Project for Infectious Diseases (2009ZX10004-101). We are grateful for the help provided by the Department of Viral Hemorrhagic Fever at the Institute of Viral Disease Control and Prevention, China CDC.

REFERENCES 1. Allwinn R, Janz B, Doerr HW. 2008. Viral gastroenteritis. An epidemiologic investigation between the period 2001-2006. Med. Klin. (Munich) 103:389 –395. 2. Anderson S, Wakeley P, Wibberley G, Webster K, Sawyer J. 2011. Development and evaluation of a Luminex multiplex serology assay to detect antibodies to bovine herpes virus 1, parainfluenza 3 virus, bovine viral diarrhoea virus, and bovine respiratory syncytial virus, with comparison to existing ELISA detection methods. J. Immunol. Methods 366:79 – 88. 3. Arnold JC. 2010. Human bocavirus in children. Pediatr. Infect. Dis. J. 29:557–558. 4. Bailey MS, et al. 2008. Viral gastroenteritis outbreaks in deployed British troops during 2002-7. J. R. Army Med. Corps. 154:156 –159. 5. Benschop K, Thomas X, Serpenti C, Molenkamp R, Wolthers K. 2008. High prevalence of human parechovirus (HPeV) genotypes in the Amsterdam region and identification of specific HPeV variants by direct genotyping of stool samples. J. Clin. Microbiol. 46:3965–3970. 6. Bharaj P, Sullender WM, Kabra SK, Broor S. 2010. Human bocavirus infection in children with acute respiratory tract infection in India. J. Med. Virol. 82:812– 816. 7. Bio-Rad Laboratories. 2010. Bio-Plex bead coupling protocol. Bio-Rad Laboratories, Hercules, CA. 8. Campe H, Hartberger C, Sing A. 2008. Role of human bocavirus infections in outbreaks of gastroenteritis. J. Clin. Virol. 43:340 –342. 9. Chieochansin T, Thongmee C, Vimolket L, Theamboonlers A, Poovorawan Y. 2008. Human bocavirus infection in children with acute gastroenteritis and healthy controls. Jpn. J. Infect. Dis. 61:479 – 481. 10. Croft H, et al. 2008. Use of Luminex xMAP-derived Bio-Plex bead-based suspension array for specific detection of PPV W and characterization of epitopes on the coat protein of the virus. J. Virol. Methods 153:203–213. 11. Deak E, et al. 2010. Utility of a Luminex-based assay for multiplexed, rapid species identification of Candida isolates from an ongoing candidemia surveillance. Can. J. Microbiol. 56:348 –351. 12. Dominguez A, Godoy P, Torner N, Cardenosa N, Martinez A. 2009. The viral gastroenteritis: a public health problem. Rev. Esp. Salud Publica 83: 679 – 687. 13. Drexler JF, et al. 2009. Novel human parechovirus from Brazil. Emerg. Infect. Dis. 15:310 –313. 14. Dunbar SA. 2006. Applications of Luminex xMAP technology for rapid, high-throughput multiplexed nucleic acid detection. Clin. Chim. Acta 363:71– 82. 15. Giordano MO, et al. 2008. Two instances of large genome profile picobirnavirus occurrence in Argentinian infants with diarrhea over a 26-year period (1977–2002). J. Infect. 56:371–375. 16. Han J. 2006. Molecular differential diagnoses of infectious diseases: is the future now? p 472–505. In Stratton C, Tang YW (ed), Advanced technologies in diagnostic microbiology. Springer, New York, NY. 17. Johansen K, et al. 2008. Norovirus strains belonging to the GII.4 genotype dominate as a cause of nosocomial outbreaks of viral gastroenteritis in Sweden 1997-2005. Arrival of new variants is associated with large nationwide epidemics. J. Clin. Virol. 42:129 –134.

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18. Karalar L, et al. 2010. Prevalence and clinical aspects of human bocavirus infection in children. Clin. Microbiol. Infect. 16:633– 639. 19. Reference deleted. 20. Liu J, et al. 2011. Multiplex reverse transcription PCR Luminex assay for detection and quantitation of viral agents of gastroenteritis. J. Clin. Virol. 50:308 –313. 21. Logan C, O’Leary JJ, O’Sullivan N. 2007. Real-time reverse transcription PCR detection of norovirus, sapovirus and astrovirus as causative agents of acute viral gastroenteritis. J. Virol. Methods 146:36 – 44. 22. Medici MC, et al. 2006. Molecular epidemiology of norovirus infections in sporadic cases of viral gastroenteritis among children in northern Italy. J. Med. Virol. 78:1486 –1492. 23. Oh DY, Gaedicke G, Schreier E. 2003. Viral agents of acute gastroenteritis in German children: prevalence and molecular diversity. J. Med. Virol. 71:82–93. 24. Oldak E, Sulik A, Rozkiewicz D, Liwoch-Nienartowicz N, Zawadzka E. 2009. Norovirus and rotavirus—two major causative agents of sporadic viral gastroenteritis in hospitalized Polish children. Adv. Med. Sci. 54:183– 186. 25. O’Neill HJ, McCaughey C, Coyle PV, Wyatt DE, Mitchell F. 2002. Clinical utility of nested multiplex RT-PCR for group F adenovirus, rotavirus and Norwalk-like viruses in acute viral gastroenteritis in children and adults. J. Clin. Virol. 25:335–343. 26. Opalka D, et al. 2003. Simultaneous quantitation of antibodies to neutralizing epitopes on virus-like particles for human papillomavirus types

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27. 28. 29. 30. 31. 32. 33. 34. 35.

36.

6, 11, 16, and 18 by a multiplexed luminex assay. Clin. Diagn. Lab. Immunol. 10:108 –115. Reference deleted. Rabenau HF, et al. 2007. Verification and validation of diagnostic laboratory tests in clinical virology. J. Clin. Virol. 40:93–98. Ramani S, Kang G. 2009. Viruses causing childhood diarrhoea in the developing world. Curr. Opin. Infect. Dis. 22:477– 482. Rasanen S, et al. 2010. Mixed viral infections causing acute gastroenteritis in children in a waterborne outbreak. Epidemiol. Infect. 138: 1227–1234. Sasaki Y, et al. 2006. Multiple viral infections and genomic divergence among noroviruses during an outbreak of acute gastroenteritis. J. Clin. Microbiol. 44:790 –797. Takao S, Hara M, Okazaki T, Suzuki K. 2011. Simultaneous multiple assay (Luminex xTAG respiratory viral panel FAST assay) efficacy in human respiratory virus detection. Kansenshogaku Zasshi 85:31–36. Ushijima H. 2009. Diagnosis and molecular epidemiology of viral gastroenteritis in the past, present and future. Uirusu 59:75–90. Reference deleted. Washington C, et al. 2010. Multiplexed Luminex xMAP assay for detection and identification of five adenovirus serotypes associated with epidemics of respiratory disease in adults. J. Clin. Microbiol. 48: 2217–2222. Zuridah H, Kirkwood CD, Bogdanovic-Sakran N, Bishop RF, Yap KL. 2010. Circulating human group A rotavirus genotypes in Malaysia. J. Med. Virol. 82:707–711.

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