Journal of Clinical Virology

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1 Journal of Clinical Virology 61 (2014) Contents lists available at ScienceDirect Journal of Clinical Virology journal homepage: Rapid and sensitive approach to simultaneous detection of genomes of hepatitis A, B, C, D and E viruses Maja Kodani, Tonya Mixson-Hayden, Jan Drobeniuc, Saleem Kamili Division of Viral Hepatitis, National Center for HIV, Hepatitis, STD and TB Prevention, Centers for Disease Control and Prevention, Atlanta, GA 30293, United States article info abstract Article history: Received 22 April 2014 Received in revised form 24 June 2014 Accepted 26 June 2014 Keywords: Simultaneous pathogen detection Real-time PCR Hepatitis Microfluidics Background: Five viruses have been etiologically associated with viral hepatitis. Nucleic acid testing (NAT) remains the gold standard for diagnosis of viremic stages of infection. NAT methodologies have been developed for all hepatitis viruses; however, a NAT-based assay that can simultaneously detect all five viruses is not available. Objectives: We designed TaqMan card-based assays for detection of HAV RNA, HBV DNA, HCV RNA, HDV RNA and HEV RNA. Study design: The performances of individual assays were evaluated on TaqMan Array Cards (TAC) for detecting five viral genomes simultaneously. Sensitivity and specificity were determined by testing 329 NAT-tested clinical specimens. Results: All NAT-positive samples for HCV (n = 32), HDV (n = 28) and HEV (n = 14) were also found positive in TAC (sensitivity, 100%). Forty-three of 46 HAV-NAT positive samples were also positive in TAC (sensitivity, 94%), while 36 of 39 HBV-NAT positive samples were positive (sensitivity, 92%). No falsepositives were detected for HBV (n = 32), HCV (n = 36), HDV (n = 30), and HEV (n = 31) NAT-negative samples (specificity 100%), while 38 of 41 HAV-NAT negative samples were negative by TAC (specificity 93%). Conclusions: TAC assay was concordant with corresponding individual NATs for hepatitis A E viral genomes and can be used for their detection simultaneously. The TAC assay has potential for use in hepatitis surveillance, for screening of donor specimens and in outbreak situations. Wider availability of TAC-ready assays may allow for customized assays, for improving acute jaundice surveillance and for other purposes for which there is need to identify multiple pathogens rapidly. Published by Elsevier B.V. 1. Background Five viruses, hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis delta virus (HDV) and hepatitis E virus (HEV), which vary widely in their natural history, genome composition, and mode of transmission [1 9], have been etiologically associated with viral hepatitis. HAV, HCV, HDV and HEV are RNA viruses while HBV is a DNA virus. HAV and HEV Abbreviations: TAC, TaqMan Array Card; NAT, nucleic acid test; HAV, hepatitis A virus; HBV, hepatitis B virus; HCV, hepatitis C virus; HDV, hepatitis D virus; HEV, hepatitis E virus; HABCDE-NAs, hepatitis A, B, C, D and E nucleic acids; LOD, limit of detection; IU, international units; GE, genome equivalents. Corresponding author at: Division of Viral Hepatitis, National Center for HIV, Hepatitis, STD, and TB Prevention, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30329, United States. Tel.: address: mkodani@cdc.gov (M. Kodani). infections, which are primarily enterically transmitted, are mostly self-resolving but can lead to acute liver failure, especially in very young children and in pregnant women, respectively [8]. Hepatitis viruses B, C, and D, which are transmitted parenterally, can lead to chronic liver disease and hepatocellular carcinoma [2 4]. HEV can also be chronic, especially in immunocompromised persons. Since viral hepatitides are clinically indistinguishable, disparate testing algorithms need to be employed to determine the exact etiology of infection. Nucleic acid testing (NAT) remains the gold standard for diagnosis of active and viremic stages of infection. Several assays, including those approved by the Food and Drug Administration (FDA), are commercially available for the detection of HBV DNA and HCV RNA. Although such assays for detecting HAV, HEV and HDV RNAs are not available, in-house methodologies for their detection and quantification have been developed. However, a NAT-based assay that can detect all five hepatitis viruses in the same format and under identical experimental conditions and by one sample preparation step has not been reported /Published by Elsevier B.V.

2 M. Kodani et al. / Journal of Clinical Virology 61 (2014) Table 1 Commercially available genotype panels and standards used in this study. Virus Name Code/reference number Institution/vendor HAV HAV RNA Working Reagent for Nucleic Acid Amplification 01/488 NIBSC, South Mimms, United Kingdom HBV AcroMetrix HBV Panel Life Technologies, Grand Island, NY, USA HBV DNA Genotype Performance Panel PHD201(M) SeraCare Life Sciences, Milford, MA, USA HCV AcroMetrix HCV Panel Life Technologies, Grand Island, NY, USA HCV RNA Genotype Performance Panel PHW202 SeraCare Life Sciences, Milford, MA, USA HEV WHO International Standard for Hepatitis E Virus RNA Nucleic Acid Amplification Techniques (NAT)-Based Assays 6329/10 Paul-Ehrlich-Institut, Langen, Germany 2. Objectives In this study, we report the use of TaqMan Array Cards (TAC, Life Technologies, Grand Island, NY), formerly known as TaqMan Low Density Array (TLDA) to facilitate rapid and simultaneous detection of HAV RNA, HBV DNA, HCV RNA, HDV RNA and HEV RNA. TAC is a set of 384 wells each well of 1 l capacity, 48 wells of which are interconnected on a plastic array card to one central port that can accommodate one clinical specimen. Each card contains eight identical ports, providing a platform to test up to eight clinical specimens simultaneously by real-time PCR. TAC technology has successfully been applied to detect etiologic agents of other syndromes, including respiratory infections [10 12], diarrhea [13], biological threat-related syndromes [14,15], and neonatal sepsis [12]. TAC-ready assays provide a potential for building modular diagnostic platforms for acute and chronic viral hepatitis, jaundice syndromic surveillance, screening of blood borne pathogens in donor blood or organs, and screening of at-risk populations including intravenous drug users and hemodialysis patients. 3. Study design 3.1. Standards, panels, and clinical specimens Table 1 summarizes all standards and genotyping panels used in this study. In addition, 329 serum specimens previously tested in our laboratory by validated assays were used. This clinical panel included samples positive for HAV RNA (n = 46), HBV DNA (n = 39), HCV RNA (n = 32), HDV RNA (n = 28) and HEV RNA (n = 14) and control samples negative for HAV RNA (n = 41), HBV DNA (n = 32), HCV RNA (n = 36), HDV RNA (n = 30), and HEV RNA (n = 31) Nucleic acid extraction Total nucleic acids (TNAs) were extracted from all samples using the MagNA Pure LC 2.0 extraction instrument and MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche, Indianapolis, IN). All extractions were performed using 200 l of serum according to the manufacturer s protocols; TNAs were eluted in 50 l of the elution buffer, aliquoted and stored at 80 C until use Assay design and PCR amplification Quantitative RT-PCR assays for detection of HAV RNA, HBV DNA, HCV RNA, HDV RNA and HEV RNA, hereinafter referred to as HABCDE-NAs, we first designed, using TAC-compatible chemistry and identical cycling conditions. In the initial evaluation, primers and probes routinely used in our laboratory for HABCDE-NAs PCR amplification [16 18] were transferred into the TAC-compatible buffer system and PCR cycling conditions, and compared to the original assay for their performance characteristics. Real-time RT-PCR reactions were performed using the ABI 7500 real-time PCR instrument using the AgPath-ID One-Step kit (Life Technologies) following the manufacturer-recommended protocols. All reactions were performed in 96-well plates in 25 l reaction volumes, each well containing 1 RT-PCR enzyme mix, 1 RT-PCR buffer, variable concentrations of primers and probes (Table 2), and 5 l of TNA extract. The following cycling conditions were used: initially 45 C for 10 min and 94 C for 10 min, followed by 45 cycles of 95 C for 30 s and 60 C for 1 min. Primers and probes used for amplification and detection of HABCDE-NAs are shown in Table 2. All the primers and probes were analyzed by BLAST [19] to ensure specificity and to determine that their Tm values were matched with the ideal 60 C for primers and 70 C for probes. Since identical chemistry and PCR cycling conditions are a requirement for all assays to be concurrently run on TAC, primer and probe concentrations were adjusted in small increments to identify the combination conferring the highest specificity and the lowest limit of detection. Once the most optimal conditions were established in the individual real-time RT-PCR assays, the HABCDE-NA assays were run on TAC in triplicate wells. In addition, two experimental controls were used in duplicate: (1) an internal positive control (IPC2) for checking the card performance; and (2) a ribonuclear protein 3 (RNP3) control, for nucleic acids extraction [10]. These assays did not utilize all the available wells on the card since the purpose of this study was to develop hepatitis assays compatible with the TAC format. Each card has eight different ports, which were utilized to run six specimens in addition to the controls. A synthetic positive control for HABCDE-NAs, described below, was used as a positive control in all runs Positive control transcript A positive control transcript was developed to encompass detection of HABCDE-NAs as described previously [18,20]. Briefly, a synthetic gene was designed containing the forward primer sequence, the probe sequence, and the reverse complement of the reverse primer sequence for HABCDE-NAs and RNP3 targets, followed by the reverse complement of the SP6 promoter sequence. This construct was synthesized and cloned into pidtsmart Amp vector (IDT, Coralville, IA). For use as a control, transcripts were generated from the plasmid construct and purified using the SP6 MEGAscript and MEGAclear kits according to the manufacturer s instructions (Life Technologies); this preparation contained 2.53x10 15 copies/ml based on the NanoDrop spectrophotometer (Thermo Scientific, Waltham, MA) and the Endmemo software ( The transcript preparation was serially diluted from the 10 8 copies/ml dilution in 1 TE buffer containing 50 ng/ml of carrier RNA, aliquoted and stored at 80 C until use.

3 262 M. Kodani et al. / Journal of Clinical Virology 61 (2014) Table 2 TaqMan qrt-pcr assay design parameters for HAV RNA, HBV DNA, HCV RNA, HDV RNA and HEV RNA. Virus Amplicon size Sequence (5 3 ) Function Conc. (nm) a Target Reference HAV 107 GGG TGA AAC CTC TTA GGC TAA TAC Forward Primer UTR This study TCC TCC GGC GTT GAA TG Reverse Primer 500 CAC CAA TAT CCG CCG CTG TTA CCC TAT CCA Probe 100 HBV 87 TGT CCT GGY TAT CGC TGG AT Forward Primer 500 X This study AAG AAC CAA YAA GAA GAT GAG Reverse Primer 500 TGC GGC GTT TTA TCA TMT T CC TCT TCA T Probe 200 HCV 95 CTA GCC GAG TAG YGT TGG GT Forward Primer 100 Core protein This study CAT GTT GCA CGG TCT ACG AG Reverse Primer 100 CTC GCA AGC ACC CTA TCA GGC AGT AC Probe 50 HDV 93 TCT CCC TTW GCC ATC MGA G Forward Primer 600 Upstream from the HDAg [18] TCC TCT TCG GGT CGG Reverse Primer 600 CYC GCG GTC CGW CCT GGG C Probe 200 HEV 70 GGT GGT TTC TGG GGT GAC Forward Primer 250 Capsid protein [16,17] AGG GGT TGG TTG GAT GAA Reverse Primer 250 TGA TTC TCA GCC CTT CGC Probe 100 a Both individual and TAC assays have the same concentrations of primers and probes. Table 3 Characteristics of TAC and corresponding individual hepatitis assays. Assay TAC LOD Individual assay LOD Slope Linear range Source of material HAV 250 ge/ml 125 ge/ml logs WHO standard HBV 500 IU/ml 100 IU/ml logs AcroMetrix standard HCV 500 IU/ml 100 IU/ml logs AcroMetrix standard HDV 1000 ge/ml 1000 ge/ml logs Transcript (in-house) HEV 2500 IU/ml 250 IU/ml logs WHO standard Table 4 TAC precision measurements based on positive control transcript data generated from 31 cards. Assay Assay (n a ) Minimum Ct Maximum Ct Average [95% CI] Standard deviation HAV [ ] HBV [ ] HCV [ ] HDV [ ] HEV [ ] RNP [ ] IPC [ ] a If assay was present on the card in triplicate, 93 data points were used in calculations. If assay was present in duplicate on the cards, 62 data points were used in calculations TaqMan array card protocol TAC assays were run on the ViiA7 instrument (Life Technologies) using the AgPath-ID One-Step Kit according to the manufacturer s instructions (Life Technologies). The TAC setup was identical to the individual reactions, with the exception of volume: 40 l of nucleic acid extract were used per 100 l reaction. Upon loading, the cards were briefly centrifuged twice, sealed and loaded into the thermal cycler. The optimized cycling conditions as described above were used to run the cards. For the purposes of this study, samples were considered positive if any Ct values, up to 45, were obtained. The reproducibility of the assay on cards was determined based on the average cycle threshold (Ct) values for 31 cards, which included 93 data points for HABCDE-NA assays each, and 62 data points for two controls, RNP3 and IPC2 assays each. The limit of detection of hepatitis TAC assay was determined using serial dilutions of standard material for HABCDE-NAs. To verify if any of the viral genomes would interfere with the detection of other viral genomes in the TAC assay, we created a set of mixed samples each spiked with one to five viruses. Finally, the assays were evaluated on 329 clinical specimens. 4. Results The characteristics of all five individual assays, including limit of detection (LOD), slope and linear range are shown in Table 3 while the characteristics of the TAC are shown in Tables 3 and 4. The precision of hepatitis TAC, as determined by the positive control transcript, indicated the amplification reproducibility of 100%. Furthermore, the Ct values of HABCDE-NAs assays were highly reproducible with tight 95% confidence intervals and low standard deviation (Table 4). The lower limit of detection for HABCDE-NAs assays was compared between TAC and its corresponding individual assays (Table 3). The HAV TAC assay was half a log 10 less sensitive than its corresponding individual assay; the HBV and HCV TAC assays were one fifth of a log 10 less sensitive than their corresponding individual assays; the HDV TAC assay was as sensitive as its corresponding individual assay; and the HEV TAC assay was 1 log 10 less sensitive than its corresponding individual assay. Amplification and detection of individual viral genomes was not affected by the presence of nucleic acids of any other hepatitis viruses (Table 5). The range of Ct values obtained for amplification of HABCDE-NAs was within the equivalent of one log 10, regardless of the presence of one, two, three, four or five hepatitis viruses in

4 M. Kodani et al. / Journal of Clinical Virology 61 (2014) Table 5 Performance of TAC assay in the amplification of individual viruses in the presence or absence of other hepatitis viruses. Number of viruses in the mix TAC performance represented by Ct values HAV HBV HCV HDV HEV N b Ave Ct b SD b N Ave Ct SD N Ave Ct SD N Ave Ct SD N Ave Ct SD a a In one case, one out of three reactions was negative. b N = number of RT-PCR reactions, Ave Ct = average crossing threshold value, SD = standard deviation. Table 6 Analytical sensitivity and specificity of TAC assays. Target True positives False negatives % sensitivity (95% CI) True negatives False positives % specificity (95% CI) % overall concordance HAV (86 100) (85 100) 93 HBV (84 100) ( ) 96 HCV ( ) ( ) 100 HDV ( ) ( ) 100 HEV ( ) ( ) 100 Total (93 99) (96 100) 97 the reaction, thus ruling out any interference of viral genomes of more than one hepatitis virus in the same sample. Based on the testing of 329 serum specimens, specificity and sensitivity were determined for hepatitis TAC assays (Table 6). Overall, considering the two- to ten-fold drop in the limit of detection, sensitivity of TAC assays remained high, ranging from 92% (HBV) to 100% (HCV, HDV and HEV). The specificity of the TAC assays was 93% for HAV and 100% for HBV, HCV, HDV, and HEV (Table 6). 5. Discussion Real time RT-PCR assays for the detection of hepatitis viruses A, B, C, D and E, adapted to the same chemistry and cycling conditions were placed on a TAC to establish a NAT test that can simultaneously detect the genomes of all the five viruses. To the best of our knowledge, this is the first test that has the potential of being used for HABCDE-NAs for serum NAT. The hepatitis TAC assay can screen human serum for the presence of any of the five hepatitis viruses simultaneously. The assay can also detect co-infections with more than one virus without affecting sensitivity or specificity. Another advantage of this test is that it uses 200 L of serum to test for the presence of five pathogens, while more than 1 ml per assay may be needed for FDA-approved individual PCR assays. The time taken for the completion of the assay from including nucleic acid extraction, TAC set-up, run and data interpretation is four hours. Although the sample is processed and loaded only once, the microfluidic technology allows for distribution of the sample into individual real-time RT- PCR reactions. In addition, the ViiA7 instrument used to run TAC assay can also be used for other molecular applications, such as 96-well and 384-well real-time PCR. The TAC cards do not need require storage at subzero temperatures, have a long shelf life up to 2 years at 4 C and can be shipped at room temperature thus not incurring undue shipping costs. The extended stability of the cards allows for simplified batch production and quality assurance. Another advantage is that additional assays can be added to the hepatitis panel without the need to re-validate the hepatitis assays, use more sample or increase the time required to generate results. Using an RT-PCR buffer system on TAC, DNA and RNA targets from virus and bacteria can be amplified in the same reaction, which is a great advantage when building syndromic panels using a combination of RNA and DNA viruses and bacteria. In the recent years, a few TAC panels were evaluated, providing a library of assays that can be mixed and matched, potentially creating even more panels with addition of a few new viral or bacterial targets [10 15]. Collectively, these panels tested a variety of clinical samples for suitability in TAC analysis, including nasopharyngeal/oropharyngeal swabs in viral transport media [10 12], bacterial cultures [14], whole blood [12,15], stool suspensions [13], and serum (this study). Any biological material could potentially be considered for TAC analysis assuming appropriate total nucleic acid extraction procedures are optimized and validated prior to specific panel development. Following this initial optimization and provision of good quality total nucleic acids from any specimen type, TAC assay results should not be heavily dependent on the extraction method. The hepatitis TAC assay has certain limitations. First, it was only evaluated as a qualitative assay. Quantitation may be possible since output is in Ct values, but running a standard curve on every card is not feasible. Further studies are needed to establish a quantitation method feasible with the TAC format. Second, it has lower sensitivity compared to corresponding individual assays. As previously reported for other TAC panels [10,13 15], the analytical limit of detection for our assays was about one log 10 lower than conventional protocols. However, the clinical significance of this lowered analytical sensitivity should not have a significant impact on the performance, since the overall sensitivity of hepatitis TAC determined on clinical specimens was 96% [95% CI: 93 99]. We have presented data showing that the hepatitis TAC assay has great potential for simplifying laboratory testing of viral hepatitides. It was not developed to replace current hepatitis testing algorithms; instead, it was created to address a need for rapid testing in situations where the traditional testing may be onerous and expensive, such as global health and disease surveillance and screening of other populations, where only a small amount of sample is available. Additionally, potential expansion of the hepatitis panel to include other pathogens, such as human immunodeficiency virus and sexually transmitted agents, may be useful in donor blood and organ testing, and in antenatal testing of pregnant women.

5 264 M. Kodani et al. / Journal of Clinical Virology 61 (2014) Funding Internal CDC funding. Competing interests The authors declare no competing interests. Ethical approval Not required. Conflict of interest The authors declare no conflict of interest. Acknowledgements We would like to thank Dr. Dean Erdman and Brett Whitaker for letting us use their laboratory for some of our experiments. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention. References [1] Martin A, Lemon SM. Hepatitis A virus: from discovery to vaccines. Hepatology 2006;43:S [2] Kao JH. Molecular epidemiology of hepatitis B virus. Korean J Intern Med 2011;26: [3] Hajarizadeh B, Grebely J, Dore GJ. Epidemiology and natural history of HCV infection. Nat Rev Gastroenterol Hepatol 2013;10: [4] Ward JW. The epidemiology of chronic hepatitis C and one-time hepatitis C virus testing of persons born during 1945 to 1965 in the United States. Clin Liver Dis 2013;17:1 11. [5] Kim CW, Chang KM. Hepatitis C virus: virology and life cycle. Clin Mol Hepatol 2013;19: [6] Niro GA, Smedile A. Current concept in the pathophysiology of hepatitis delta infection. Curr Infect Dis Rep 2012;14:9 14. [7] Hughes SA, Wedemeyer H, Harrison PM. Hepatitis delta virus. Lancet 2011;378: [8] Aggarwal R, Jameel S. Hepatitis E. Hepatology 2011;54: [9] Panda SK, Thakral D, Rehman S. Hepatitis E virus. Rev Med Virol 2007;17: [10] Kodani M, Yang G, Conklin LM, Travis TC, Whitney CG, Anderson LJ, et al. Application of TaqMan low-density arrays for simultaneous detection of multiple respiratory pathogens. J Clin Microbiol 2011;49: [11] Weinberg GA, Schnabel KC, Erdman DD, Prill MM, Iwane MK, Shelley LM, et al. Field evaluation of TaqMan Array Card (TAC) for the simultaneous detection of multiple respiratory viruses in children with acute respiratory infection. J Clin Virol 2013;57: [12] Diaz MH, Waller JL, Napoliello RA, Islam MS, Wolff BJ, Burken DJ, et al. Optimization of multiple pathogen detection using the TaqMan Array Card: application for a population-based study of neonatal infection. PLOS ONE 2013;8: e [13] Liu J, Gratz J, Amour C, Kibiki G, Becker S, Janaki L, et al. A laboratory-developed TaqMan Array Card for simultaneous detection of 19 enteropathogens. J Clin Microbiol 2013;51: [14] Rachwal PA, Rose HL, Cox V, Lukaszewski RA, Murch AL, Weller SA. The potential of TaqMan Array Cards for detection of multiple biological agents by real-time PCR. PLOS ONE 2012;7:e [15] Weller SA, Cox V, Essex-Lopresti A, Hartley MG, Parsons TM, Rachwal PA, et al. Evaluation of two multiplex real-time PCR screening capabilities for the detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis in blood samples generated from murine infection models. J Med Microbiol 2012;61: [16] Garson JA, Ferns RB, Grant PR, Ijaz S, Nastouli E, Szypulska R, et al. Minor groove binder modification of widely used TaqMan probe for hepatitis E virus reduces risk of false negative real-time PCR results. J Virol Methods 2012;186: [17] Jothikumar N, Cromeans TL, Robertson BH, Meng XJ, Hill VR. A broadly reactive one-step real-time RT-PCR assay for rapid and sensitive detection of hepatitis E virus. J Virol Methods 2006;131: [18] Kodani M, Martin A, Mixson-Hayden T, Drobeniuc J, Gish RR, Kamili S. One-step real-time PCR assay for detection and quantitation of hepatitis D virus RNA. J Virol Methods 2013;193: [19] Altschul SF, Gertz EM, Agarwala R, Schaffer AA, Yu YK. PSI-BLAST pseudocounts and the minimum description length principle. Nucleic Acids Res 2009;37: [20] Kodani M, Winchell JM. Engineered combined-positive-control template for real-time reverse transcription-pcr in multiple-pathogen-detection assays. J Clin Microbiol 2012;50: