The Journal of Molecular Diagnostics, Vol. 16, No. 1, January 2014
jmd.amjpathol.org
Multiplex Screening for Blood-Borne Viral, Bacterial, and Protozoan Parasites using an OpenArray Platform Elena Grigorenko,* Carolyn Fisher,y Sunali Patel,* Caren Chancey,y Maria Rios,y Hira L. Nakhasi,y and Robert C. Duncany From Life Technologies Corp.,* South San Francisco, California; and the Division of Emerging and Transfusion Transmitted Diseases,y Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland Accepted for publication August 22, 2013. Address correspondence to Robert C. Duncan, Ph.D., 1401 Rockville Pike, HFM310, Rockville, MD 20852. E-mail: [email protected].
The use of nucleic acid tests for detection of pathogens has improved the safety of blood products. However, ongoing pathogen emergence demonstrates a need for development of devices testing for multiple pathogens simultaneously. One approach combines two proven technologies: Taqman chemistry for target identification and quantification and the OpenArray nanofluidic real-time PCR platform for spatial multiplexing of assays. A panel of Taqman assays was developed to detect nine blood-borne pathogens (BBPs): four viral, two bacterial, and three protozoan parasites. The custom BBP OpenArray plate with 18 assays was tested for specificity and analytical sensitivity for nucleic acid from each purified pathogen and with pathogen-spiked human blood and plasma samples. For most targets, the limits of detection (10 to 10,000 copies/mL) were comparable with existing real-time platforms. The testing of the BBP OpenArray with pathogen-spiked coded human plasma or blood samples and negative control specimens demonstrated no false-positive results among the samples tested and correctly identified pathogens with the lowest concentration detected ranging from 10 cells/mL (Trypanosoma cruzi) to 10,000 cells/mL (Escherichia coli). These results represent a proof of concept that indicated the BBP OpenArray platform in combination with Taqman chemistry may provide a multiplex real-time PCR pathogen detection method that points the way for a next-generation platform for infectious disease testing in blood. (J Mol Diagn 2014, 16: 136e144; http://dx.doi.org/10.1016/j.jmoldx.2013.08.002)
Testing of donations for blood-borne pathogens (BPPs) has reduced significantly the risk of transfusion transmission of such agents. However, the current number of pathogen tests and the increasing number of emerging pathogens that can affect blood safety results in an increased number of screening tests required before release of the blood and blood products. The use of a single pathogen assay for each agent is burdensome and costly and requires volumes of specimens that risk exceeding the maximum collection volume allowed for testing. Devices that allow simultaneous testing for multiple pathogens (multiplex testing) can potentially streamline blood donation testing. To date, the highest number of pathogens that can be assayed in one multiplex test licensed for blood screening is five [HIV-1 group M and group O, HIV-2, hepatitis C virus (HCV), and hepatitis B virus (HBV)].1 Current multiplex tests require subsequent discriminatory assays to identify which pathogen produced the reactive test result. An improved technology using real-time nucleic acid tests Copyright ª 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jmoldx.2013.08.002
(NATs) that promise to deliver discrimination simultaneously with detection is available in Europe but not the United States (TaqScreen MPX Test, version 2.0; Roche, Roche Molecular Diagnostics, Pleasanton, CA) and other devices that combine amplification with microarray hybridization have also been proposed.2 New technology needs to be developed that can achieve in a single assay both higher levels of multiplicity and simultaneous discrimination of the reactive agent. Application of novel and emerging technologies offers the prospect to develop the next generation of superior performance tests that Supported in part by an internal FDA grant under the Modernizing Science Program. Disclosures: E.G. and S.P. are employees of Life Technologies Corp., the manufacturer of the OpenArray platform. The comments in this publication are an informal communication and represent the authors’ own best judgments. These comments do not bind or obligate the FDA. The devices described in this publication have not been formally cleared or approved by the FDA for the uses discussed herein.
Multiplex Blood-Borne Pathogen Screening will detect an increasing number of transfusion-transmittable agents in multiplex format with high sensitivity and specificity, robustness, and adaptability to accomplish detection of new agents. To date, NATs have only been applied to viral pathogens, namely, HIV, HCV, HBV, and West Nile virus (WNV) using plasma specimens. Testing of plasma specimens would be substantially improved if all of these viral NAT assays could be performed on a single device with simultaneous detection and discrimination. Further, no Food and Drug Administrationeapproved NAT is being performed for blood donor screening of whole blood specimens. Nucleic acid detection of bacteria and protozoan parasites, such as Plasmodium, Trypanosoma cruzi, and Leishmania, that would be associated with whole blood specimens would make a significant contribution to blood safety if available on a multiplex assay of sufficient sensitivity. A multiplex approach can be achieved by using assays with different reporters combined in the same reaction well or by using high densities of physically independent PCR wells. However, both approaches have challenges. The presence of multiple primers may lead to cross-hybridization with each other and the possibility of mispriming with other templates, so optimization is required for any new primer sets in multiplex PCR. As for the other approach, stringent fluidic isolation between adjacent PCR reaction wells is necessary to prevent cross-contamination during loading and temperature cycling on any PCR platform with a high density of independent wells. Recent advancements in high-throughput realtime PCR technologies, such as the OpenArray platform (Life Technologies Corp., Carlsbad, CA), address these disadvantages by assembling selected primer and probe sets in spatially isolated reaction wells and combine the sensitivity and specificity of quantitative PCR with the highthroughput performance of microarrays to achieve largescale screening of pathogens.3e5 In this study, we evaluated a custom BBP OpenArray panel composed of primer and probe sets developed by scientists from both our institutions and published sources.6e8 We determined its limit of detection for four viral pathogens in human plasma specimens simultaneously and five bacterial and protozoan pathogens in human blood specimens simultaneously. We also evaluated the custom BBP OpenArray’s ability to correctly identify coded specimens at the limit of detection.
Materials and Methods Pathogen Culture and Specimen Preparation HIV, HCV, and HBV were prepared from Center for Biologics Evaluation and Research (CBER) lot release panel members that were composed of human plasma specimens from infected individuals or infected cell culture, diluted with pooled human plasma defibrinated and delipidated, and tested negative for common BBPs (base matrix) to 500 or 100
The Journal of Molecular Diagnostics
-
jmd.amjpathol.org
copies/mL, inactivated, and stored in 1-mL vials at 80 C. The viral load of the frozen vials was extensively validated, as previously described for HIV9,10 and HCV.11 The HBV panel members were derived from source material that was a specimen obtained from a blood donor in the United States [HBV DNA, genotype A, serotype adw2; Food and Drug Administration (FDA) identification No. 723534]. The panel members were diluted in base matrix and tested to contain 100 and 500 DNA copies/mL (Steve Kerby, FDA, unpublished data). The lot release panel members used were obtained directly from the Product Testing Section, Division of Emerging and Transfusion Transmitted Diseases, CBER, FDA. WNV was cultured on Vero cells,12 and the supernatant was collected and viral RNA quantified by both plaqueforming units per milliliter determined by plaque assay13 and copies per milliliter in limiting dilution and a TaqMan assay.14,15 Two bacterial species, Escherichia coli and Yersinia enterocolitica, were cultured in Super Broth (Quality Biological Inc., Gaithersburg, MD) and quantified by measuring optical density at 600 nm and converting to cells per milliliter by the formula 5.0 108 cells/mL Z 1 absorbance unit with the SmartSpec 3000 spectrophotometer (BioRad Laboratories Inc., Hercules, CA). Leishmania donovani 1S2D promastigote form was cultured as previously described16 and quantified with a Beckman/Coulter particle counter. T. cruzi Columbiana strain parasites were cultured as epimastigotes as previously described17 and quantified with a Beckman/Coulter particle counter. Plasmodium falciparum was cultured as previously described18 and quantified by microscopic determination of the percentage of red blood cells infected and determination of the number of red blood cells per milliliter with a Beckman/ Coulter particle counter, then the Plasmodium cells were calculated by the product of these two numbers.
Spiking For HIV, HCV, and HBV, the 1-mL aliquoted lot release panel members were used directly as test specimens without further dilution. WNV-spiked specimens were prepared (under biosafety level 3 containment) from live WNV cell culture stock at a concentration of 1010 copies/mL as determined by various methods in collaborative studies. An intermediate dilution of 1:1000 containing 107 copies/mL of WNV was prepared in PBS, followed by a second intermediate dilution of 1:100 in human plasma to have 105 copies/mL, which was further subjected to a 10-fold serial dilution in 1-mL aliquots of human plasma to prepare the testing specimens that contained 104, 103, and 102 copies/ mL. E. coli, Y. enterocolitica, L. donovani, T. cruzi, and P. falciparum were similarly diluted and spiked into whole heparinized human blood. The blood and plasma were obtained from deidentified donors to the research program at the Department of Transfusion Medicine, National Institutes of Health, under an approved protocol (principal investigator R.C.D.).
137
Grigorenko et al Table 1
Assays for Plasma and Whole BBPs Found in Public Sources or Available on Request Amplicon
Species
Target gene
Assay name
Accession No.*
Coordinates
Size (bp)
Reference
HIV HIV HIV HCV Leishmania Leishmania T. cruzi T. cruzi
GAG GAG GAG 3’UTR X-tail Minicircle 18S rRNA Minisatellites Minisatellites
HIV-1A HIV-1B HIV-1C HCV-A LEI-1 LEI-2 TCF-1 TCF-2
JN417241.1 JN417241.1 JX973372 EU835523.1 AB678348.1 FR799614.1 AY520036y AY520036y
684-797 677-755 724-794 3-56 10-125 1020493- 1020707 26-190 26-187
113 78 70 53 115 214 164 161
I. Hewlett, FDA, unpublished data I. Hewlett, FDA, unpublished data R.C.D., FDA, unpublished data 6 7 R.C.D., FDA, unpublished data 8 R.C.D., FDA, unpublished data
*Accession numbers are for GenBank (http://www.ncbi.nlm.nih.gov/genbank). y Two assays only differ by probe sequence. bp, base pair.
Nucleic Acid Extraction Nucleic acid from 140 mL of WNV-spiked plasma was extracted using the QIAamp Viral Mini Kit (Qiagen, Valencia, CA) according to the manufacturer protocol and RNA eluted twice with 100 mL of elution buffer each elution into the same tube. Nucleic acid from 1 mL of all other plasma specimens was extracted with the QIAamp MinElute Virus Spin Kit (Qiagen) according to the manufacturer protocol and eluted in 180 mL of elution buffer. DNA from 1-mL whole blood specimens was extracted with the QIAamp DNA Blood Mini Kit (Qiagen) according to the manufacturer protocol and eluted in 180 mL of elution buffer.
Primer Design and Selection The panel for detection of BBPs consisted of primers and Taqman probes for viral, bacterial, and parasitic targets (Tables 1 and 2) designed for this study, published in the literature or commercially available from Life Technologies Corp. A proprietary algorithm was applied for a subset of Taqman assays designed by Life Technologies Corp. This algorithm evaluates a set of optimal assays, considering criteria of melting temperature and nucleotide composition of primerpair combinations. The algorithm selects the assays with highest specificity, based on nucleic acid sequence comparison of assay primers and probes with genomic sequences from other closely related species. Based on sequence comparison, the assay with the highest mismatch score with other Table 2
organisms was chosen, which minimizes the possibility of generation of false-positive results during the testing. Primers and probes designed by the FDA laboratory as part of this study were selected from genomic sequence using MacVector software version 11.1.2 (MacVector, Inc., Cary, NC), avoiding hairpin loops, primer duplexes, and optimizing melting temperatures.
Reverse Transcription Reaction and Preamplification Nucleic acid solution (10 mL) extracted from plasma samples was reverse transcribed using the High Capacity Reverse Transcription Kit (Life Technologies Corp., P/N 4368814) with random hexamer primers or gene-specific primers (pool of all assay primers diluted 1:5 relative to the concentration in the PCR reaction), in a total volume of 20 mL, incubated at 37 C for 2 hours per manufacturer protocol. To increase sensitivity for targets present at low concentration, targetspecific preamplification was performed. A 10-microliter sample of cDNA was mixed with 10 mL of a mixed pool of PCR assay oligonucleotides at 180 nmol/L-50 nmol/L primer pair-probe concentration and 20 mL of Taqman preamplification master mix (Life Technologies Corp., P/N 4384267). The preamplification reaction was performed on an ABI9700 at the following cycling conditions: single cycle (95 C for 10 minutes), 18 cycles (95 C for 15 seconds and 60 C for 1 minute), and one cycle (95 C for 5 minutes). Preamplification products were diluted 1:10 in 0.1 TE buffer and were used on the BBP OpenArray platform.
Commercial Taqman Assays Available from Life Technologies Corp.
Assay name
LT assay No.
Organism
Gene
Target accession No.
HCV-B HBV-A HBV-B WNV-A WNV-B PLA-1 PLA-2 GNEG-1 GNEG-2
Pa03453408_s1 Pa03453405_s1 Pa03453406_s1 Pa04329496_s1 Pa04329497_s1 Pa04329498_s1 Pa04329499_s1 Pa04329500_s1 Pa04329501_s1
HCV HBV HBV WNV WNV Plasmodium species Plasmodium species Gram-negative bacteria Gram-negative bacteria
50 UTR P, S P, X 30 UTR 30 UTR Mitochondrial rRNA Mitochondrial rRNA gapA gapA
AF009606 X04615 X04615 AF202541.1 AF533540.1 GQ355486.1 AY791633.1 X02662.1 X02662.1
138
jmd.amjpathol.org
-
The Journal of Molecular Diagnostics
Multiplex Blood-Borne Pathogen Screening Table 3
BBP OpenArray Results With Virus-Spiked Plasma Specimens Assay 1
Pathogen (copies/mL) HIV (500) HCV (500) HBV (500) WNV (100) WNV (1000) WNV (10,000)
Assay 2
Assay 3
Name
Mean Cq (95% CI)
No. of positive/total through-holes Name
Mean Cq (95% CI)
HIV-1A HCV-A HBV-A WNV-A WNV-A WNV-A
14.3 NA 18.5 21.3 19.2 14.7
24/24 0/24 24/24 24/24 24/24 24/24
17.4 19.2 19.4 NA 25.8 20.0
(0.1) (0.05) (0.06) (0.05) (0.05)
HIV-1B HCV-B HBV-B WNV-B WNV-B WNV-B
No. of positive/ total throughholes Name
(0.1) 12/24 (0.04) 24/24 (0.04) 24/24 0/24 (0.27) 24/24 (0.07) 24/24
Mean Cq (95% CI)
No. of positive/total through-holes
HIV-1C 14.2 (0.2) 5/24
NA, not applicable.
Real-Time PCR on the BBP OpenArray Platform The OpenArray technology is based on a metal plate the size of a microscope slide that has been photolithographically patterned and etched to form a rectilinear array of 3072 throughholes, organized in 48 subarrays with 64 through-holes each. Each through-hole is loaded with individual Taqman assays and contains 33 nL of PCR mixture. Previous work4 has shown that the PCR assay performance in the nanoplates is equivalent to the same assay in microplates but with a >150-fold lower reaction volume (33-nL versus 5-mL PCR reaction volumes) and with the ability to profile multiple targets using the same sample. cDNA and genomic DNA samples were tested on custom BBP OpenArray plates containing 18 Taqman assays spotted in triplicate. Genomic DNA or cDNA (1.2 mL of each) was mixed with 1.3 mL of PCR-grade water and 2.5 mL of Taqman OpenArray real-time PCR master mix (Life Technologies Corp., P/N 4462159). Then, the reaction mixture was dispensed on a BBP OpenArray plate using the automated sample loading system Accufill (Life Technologies, Corp.). Real-time PCR occurred in an OpenArray custom computercontrolled imaging thermal cycler under software control, where imaging data for up to 9216 individual reaction wells (three plates) were collected during 40 cycles of PCR. The Table 4
postacquisition step includes calculation of quantification cycle (Cq) and the CT confidence parameter, which were used for further data analysis and in the decision tree for pathogen identification. The CT confidence parameter is calculated using Life Technologies Corp.’s proprietary algorithm, and its value reflects the quality of the amplification curve generated during PCR. The CT confidence values >300 indicate good quality of amplification. The CT confidence values of 200 through 300 are considered marginal, and each amplification curve with such CT confidence values has to be manually checked before the result can be considered valid. Reactions with CT confidence values 50% Through-holes/assay for one assay/pathogen Yes
Ct Con.30*
No
Reproducibility Some Through-holes positive for all assays/pathogen
No
Negative
Yes Specificity Cq values and Through-hole count substantially higher than all other pathogens
No
Indeterminate
Figure 1 The decision tree for interpretation of BBP OpenArray results. This flow diagram indicates the series of questions that would be asked of each assay result, then the results of each pair of assays that detect a particular pathogen to determine whether the results indicate that pathogen has been detected. The flow initiates at the upper left corner. Ct Con, Ct confidence (a measure of the quality of the amplification curve). Asterisk: One exception to this rule is made for the P. falciparum assays, which have a higher level of specificity; Cq values up to 35 are accepted to indicate the presence of that pathogen.
Yes
Retest
Pathogen Identified
random primers or a pool of gene-specific primers, then preamplified for 18 cycles with a pool of all 18 primer pairs. A 1:10 dilution of this preamplification mix was used as template in the BBP OpenArray. Two criteria were used for assessment of a standard experiment with BBP OpenArray plates: counting the number of through-holes with amplification detected for each reactive assay of the total number of through-holes used for that assay and the mean Cq values for the reactive through-holes. A series of experiments that led to the determination of the limits of detection for each virus from spiked samples are given in Table 3, where the results are reported for the three assays for HIV and two assays for the other viruses. This set of experiments suggested that on a BBP OpenArray plate, HIV and HBV could be detected below 500 copies/mL, HCV was not consistently detected at 500 copies/mL, and WNV could be detected consistently at 1000 copies/mL. This is an unexpectedly high concentration for the limit of detection for WNV and will be discussed further below. Unspiked control specimens (pathogen-free plasma) were also tested on the same OpenArray plates and showed no amplification for any assays. On the basis of comparison of amplification results for each assay, a set of criteria (mean Cq values and the CT confidence parameter, which reflects the quality of the amplification curve, reproducibility among the replicates of
The Journal of Molecular Diagnostics
-
jmd.amjpathol.org
an assay, reproducibility among the assays targeting a particular pathogen, and assay specificity) were compiled that completely distinguished a true-positive result from a true-negative result among the spiked plasma specimens with known virus concentrations or plasma alone. These criteria were expressed in a decision tree (Figure 1) that was applied to all of the replicates of each assay for each sample to reach a decision regarding the presence or absence of each pathogen in each sample. Guided by this decision tree, we proceeded to evaluate blinded specimens. To evaluate the ability of the BBP OpenArray plates to identify unknown viral specimens, plasma was spiked with measured amounts of cultured pathogens or selected CBER lot release panel members (see Materials and Methods) near the limits of detection determined above. The testing of 25 coded nucleic acid specimens, including pathogen-free plasma on BBP OpenArray plates, resulted in 16 truepositive, four true-negative, five false-negative, and no false-positive results (Table 4), which is a success rate of 80% (20/25; 95% CI, 60.9%e91.1%). Among these results were three specimens of WNV at 100 copies/mL and one at 10 copies/mL, all detected successfully, indicating the expected lower limit of detection is achievable for WNV on the BBP OpenArray plates. Most of the false-negative results occurred in the specimens with the lowest virus concentrations (100 copies/mL).
141
Grigorenko et al
Evaluation of Whole Blood Specimens
A BBP multiplex real-time PCR device has been assembled that could screen a blood specimen for four virus species, three protozoan parasite species, and a whole group of bacteria simultaneously by splitting the sample into plasma and red blood cell fractions. The tested pathogens, HBV, HCV, HIV-1, WNV, gram-negative bacteria, Leishmania, Plasmodium, and T. cruzi, are all agents that pose a significant risk for transmission in blood or blood component transfusion. These pathogens were all correctly identified in blinded testing on a BBP OpenArray platform at limits of detection of a slightly higher pathogen concentration than those observed with lower multiplicity tests that detect virus in plasma23e27 or DNA genome pathogens in blood.28e32 This study represents a demonstration of the proof of concept for an advance in multiplexing of blood donor screening and diagnostics. The extraction method using offthe-shelf column-based technology can ensure reproducibility for DNA and RNA yields from clinical specimens. In optimizing the reverse transcription step, gene-specific primers were selected because they led to generally higher cDNA yield than random primers. To achieve lower limits
of detection, an initial preamplification step was added wherein a larger volume of specimen with a pool of the primers with the same sequence as those on the BBP OpenArray platform was thermocycled 10 to 18 times. The addition of a preamplification PCR reaction to improve sensitivity increases the risk of contamination as reported by others.33 However, care in handling, separation of PCR setup and PCR product manipulation into different rooms, and monitoring each reaction with no-template control tubes eliminated false-positive results in our hands. Optimization of the BBP OpenArray benefited from extensive prior experience with microorganism detection with this platform.3,4,34 The flexibility of the BBP OpenArray platform derived from user selection of assays, which can be chosen from user designed primers and probes or from a commercial vendor. Many of the primers used in this study were used previously as an optimized assay in singletube, 384-well or 96-well plates. Some additional primers had been designed specifically for this study and underwent experimental validation for specificity and sensitivity. The choice of primer sequence also involved optimizing amplicon length, a property that must balance efficiency of amplification (shorter length) with specificity of detection (sufficient length to accommodate an optimal fluorescent hydrolysis probe). The chosen layout of the assays, each subarray with the set of 18 assays in triplicate, meant that 48 specimens could be amplified on one BBP OpenArray plate (one specimen to each subarray) with each specimen tested three times with each assay. Alternatively, a specimen could be loaded on multiple subarrays, increasing the number of replicates but decreasing the total number of specimens that could be processed per BBP OpenArray plate. This alternative was our preferred approach because the larger number of replicates enhanced the sensitivity and specificity. The BBP OpenArray performance was validated with whole human blood and plasma spiked with the agent in 1 mL volume at the specified concentration. This approach ensures that all of the inhibitors that might be present in infected human blood or plasma are present and the extraction technique is challenged to recover the pathogen genome from the volume and complexity of a natural sample. Of course, subtle differences remain in an actual infected human specimen, so consequently testing clinically derived samples will be the subject of future investigations. The eightfold level of multiplicity achieved represents a substantial improvement in streamlining blood donor screening and clinical diagnostics. However, this is only proof of the concept that the BBP OpenArray can perform detection with microorganismcontaminated blood and plasma. This platform has capacity to expand to higher levels of multiplicity. The flexibility of the platform is also a much desired asset, allowing addition or removal of assays as new agents emerge or crises, such as bioweapons, are deployed. The existence of a fully validated platform that can adapt with only minor modification is needed. The design of the BBP OpenArray may fulfill this need because each assay is performed in a separate through-
142
jmd.amjpathol.org
The pathogens targeted for detection in whole blood specimens were all DNA genome organisms, such as bacteria and protozoan parasites. Volunteer blood was spiked with cultured pathogens at known concentrations and DNA extracted, and the nucleic acid sample was tested on the BBP OpenArray plates. Multiple runs with specimens spiked at 10-fold dilutions showed reproducible and specific detection at the highest concentration but intermittent success at lower concentrations (Table 5). A preamplification step is widely used for gene expression profiling to increase sensitivity of the detection in real-time PCR when starting material is limited.21,22 To achieve reliable detection of pathogens at the lower concentrations, each whole blood specimen was preamplified with a pool of all of the assay primers and probes before loading on the BBP OpenArray plates. The criteria in the pathogen identification decision tree (Figure 1) developed for the plasma specimens were applied without modification to the assessment of the whole blood specimens. Unknown specimens were prepared by spiking cultured bacteria and protozoan parasites into whole blood at known concentrations near and below the limits of detection determined above. Twenty-three coded specimens were tested at least three times on separate OpenArray plates with 9 to 18 replicate through-holes for each assay. The decision tree was used to make a determination from the combined results of the three runs for each specimen (Table 6). Overall, among the 23 specimens, there were 22 correct calls (true-positive or true-negative results, 96% (95% CI, 79.5%e99.3%) and one incorrect call (4%). There were no false-positive results.
Discussion
-
The Journal of Molecular Diagnostics
Multiplex Blood-Borne Pathogen Screening hole with little likelihood that a new assay would interfere with an existing one. Novel technology has been applied to a recognized problem in testing blood and blood products for infectious agents. The BBP OpenArray platform customized with BBP real-time PCR assays has demonstrated a higher level of multiplicity with sensitivity and specificity, suggesting that the concept can be further developed to achieve suitability for clinical use.
13.
14.
15.
Acknowledgments We thank Jiangqin Zhao, Indira Hewlett, Stephen Kerby, Nancy Lee, Victoria Majam, Sanjai Kumar, Rana Nagarkatti, and Alain Debrabant (all from CBER, FDA, Bethesda, MD) for primer sequences and pathogen source material.
References
16.
17.
18.
1. Margaritis AR, Brown SM, Seed CR, Kiely P, D’Agostino B, Keller AJ: Comparison of two automated nucleic acid testing systems for simultaneous detection of human immunodeficiency virus and hepatitis C virus RNA and hepatitis B virus DNA. Transfusion 2007, 47:1783e1793 2. Tomioka K, Peredelchuk M, Zhu X, Arena R, Volokhov D, Selvapandiyan A, Stabler K, Mellquist-Riemenschneider J, Chizhikov V, Kaplan G, Nakhasi H, Duncan R: A multiplex polymerase chain reaction microarray assay to detect bioterror pathogens in blood. J Mol Diagn 2005, 7:486e494 3. Gonzales TK, Kulow M, Park DJ, Kaspar CW, Anklam KS, Pertzborn KM, Kerrish KD, Ivanek R, Dopfer D: A high-throughput open-array qPCR gene panel to identify, virulotype, and subtype O157 and non-O157 enterohemorrhagic Escherichia coli. Mol Cell Probes 2011, 25:222e230 4. Morrison T, Hurley J, Garcia J, Yoder K, Katz A, Roberts D, Cho J, Kanigan T, Ilyin SE, Horowitz D, Dixon JM, Brenan CJ: Nanoliter high throughput quantitative PCR. Nucleic Acids Res 2006, 34:e123 5. Slinger R, Moldovan I, Barrowman N, Chan F: Successful nanolitre real-time PCR detection of respiratory pathogens in clinical specimens. Clin Microbiol Infect 2012, 18:E286eE288 6. Drexler JF, Kupfer B, Petersen N, Grotto RM, Rodrigues SM, Grywna K, Panning M, Annan A, Silva GF, Douglas J, Koay ES, Smuts H, Netto EM, Simmonds P, Pardini MI, Roth WK, Drosten C: A novel diagnostic target in the hepatitis C virus genome. PLoS Med 2009, 6:e31 7. Nicolas L, Prina E, Lang T, Milon G: Real-time PCR for detection and quantitation of leishmania in mouse tissues. J Clin Microbiol 2002, 40: 1666e1669 8. Piron M, Fisa R, Casamitjana N, Lopez-Chejade P, Puig L, Verges M, Gascon J, Gomez i Prat J, Portus M, Sauleda S: Development of a realtime PCR assay for Trypanosoma cruzi detection in blood samples. Acta Trop 2007, 103:195e200 9. Davis C, Heath A, Best S, Hewlett I, Lelie N, Schuurman R, Holmes H: Calibration of HIV-1 working reagents for nucleic acid amplification techniques against the 1st international standard for HIV-1 RNA. J Virol Methods 2003, 107:37e44 10. Lee S, Wood O, Taffs RE, Hu J, Machuca A, Vallejo A, Hewlett I: Development and evaluation of HIV-1 subtype RNA panels for the standardization of HIV-1 NAT assays. J Virol Methods 2006, 137: 287e291 11. Saldanha J, Heath A, Lelie N, Pisani G, Nubling M, Yu M: Calibration of HCV working reagents for NAT assays against the HCV
The Journal of Molecular Diagnostics
12.
-
jmd.amjpathol.org
19.
20. 21.
22.
23.
24.
25.
26.
27.
28.
international standard: The Collaborative Study Group. Vox Sang 2000, 78:217e224 Grinev A, Daniel S, Laassri M, Chumakov K, Chizhikov V, Rios M: Microarray-based assay for the detection of genetic variations of structural genes of West Nile virus. J Virol Methods 2008, 154:27e40 Rios M, Zhang MJ, Grinev A, Srinivasan K, Daniel S, Wood O, Hewlett IK, Dayton AI: Monocytes-macrophages are a potential target in human infection with West Nile virus through blood transfusion. Transfusion 2006, 46:659e667 Rios M, Daniel S, Chancey C, Hewlett IK, Stramer SL: West Nile virus adheres to human red blood cells in whole blood. Clin Infect Dis 2007, 45:181e186 Rios M, Daniel S, Dayton AI, Wood O, Hewlett IK, Epstein JS, Caglioti S, Stramer SL: In vitro evaluation of the protective role of human antibodies to West Nile virus (WNV) produced during natural WNV infection. J Infect Dis 2008, 198:1300e1308 Debrabant A, Joshi MB, Pimenta PFP, Dwyer DM: Generation of Leishmania donovani axenic amastigotes: their growth and biological characteristics. Intl J Parasitol 2004, 34:205e217 McDaniel JP, Dvorak JA: Identification, isolation, and characterization of naturally-occurring Trypanosoma cruzi variants. Mol Biochem Parasitol 1993, 57:213e222 Mahajan B, Zheng H, Pham PT, Sedegah MY, Majam VF, Akolkar N, Rios M, Ankrah I, Madjitey P, Amoah G, Addison E, Quakyi IA, Kumar S: Polymerase chain reaction-based tests for pan-species and species-specific detection of human Plasmodium parasites. Transfusion 2012, 52:1949e1956 Sokal R, Rohlf F: Biometry: The Principles and Practice of Statistics in Biological Research. ed 2. New York, W. H. Freeman and Company, 1981 Wilson EB: Probable inference, the law of succession, and statistical inference. J Am Stat Assoc 1927, 22:209e212 Li J, Smyth P, Cahill S, Denning K, Flavin R, Aherne S, Pirotta M, Guenther SM, O’Leary JJ, Sheils O: Improved RNA quality and TaqMan Pre-amplification method (PreAmp) to enhance expression analysis from formalin fixed paraffin embedded (FFPE) materials. BMC Biotechnol 2008, 8:10 Noutsias M, Rohde M, Block A, Klippert K, Lettau O, Blunert K, Hummel M, Kuhl U, Lehmkuhl H, Hetzer R, Rauch U, Poller W, Pauschinger M, Schultheiss HP, Volk HD, Kotsch K: Preamplification techniques for real-time RT-PCR analyses of endomyocardial biopsies. BMC Mol Biol 2008, 9:3 Araujo F, Henriques I, Monteiro F, Meireles E, Cruz A, Tavares G, Mota-Miranda A: Evaluation of NucliSens-AmpliScreen methodology to detect subtypes G of HIV-1 and 4c/4d of HCV in the screening of blood donors. Transfus Clin Biol 2005, 12:331e335 Busch MP, Tobler LH, Saldanha J, Caglioti S, Shyamala V, Linnen JM, Gallarda J, Phelps B, Smith RI, Drebot M, Kleinman SH: Analytical and clinical sensitivity of West Nile virus RNA screening and supplemental assays available in 2003. Transfusion 2005, 45: 492e499 Li L, Chen PJ, Chen MH, Chak KF, Lin KS, Tsai SJ: A pilot study for screening blood donors in Taiwan by nucleic acid amplification technology: detecting occult hepatitis B virus infections and closing the serologic window period for hepatitis C virus. Transfusion 2008, 48: 1198e1206 Pichl L, Heitmann A, Herzog P, Oster J, Smets H, Schottstedt V: Magnetic bead technology in viral RNA and DNA extraction from plasma minipools. Transfusion 2005, 45:1106e1110 Pisani G, Pupella S, Marino F, Gaggioli A, Sambri V, Rossini G, Wirz M, Grazzini G: Interlaboratory study to evaluate the performance of laboratories involved in West Nile virus RNA screening of blood and blood components by nucleic acid amplification testing in Italy. Blood Transfus 2011, 9:425e429 Mary C, Faraut F, Lascombe L, Dumon H: Quantification of Leishmania infantum DNA by a real-time PCR assay with high sensitivity. J Clin Microbiol 2004, 42:5249e5255
143
Grigorenko et al 29. Najdenski H, Heyndrickx M, Herman L, Werbrouck H, Van Coillie E: Quantification of Yersinia enterocolitica in raw milk using qPCR. Vet Microbiol 2012, 160:428e434 30. Patel P, Garson JA, Tettmar KI, Ancliff S, McDonald C, Pitt T, Coelho J, Tedder RS: Development of an ethidium monoazideenhanced internally controlled universal 16S rDNA real-time polymerase chain reaction assay for detection of bacterial contamination in platelet concentrates. Transfusion 2012, 52:1423e1432 31. Schachterle SE, Mtove G, Levens JP, Clemens EG, Shi L, Raj A, Munoz B, Reller ME, West S, Dumler JS, Sullivan D: Prevalence and density-related concordance of three diagnostic tests for malaria in a region of Tanzania with hypoendemic malaria. J Clin Microbiol 2011, 49:3885e3891 32. Schijman AG, Bisio M, Orellana L, Sued M, Duffy T, Mejia Jaramillo AM, Cura C, Auter F, Veron V, Qvarnstrom Y, Deborggraeve S, Hijar G, Zulantay I, Lucero RH, Velazquez E,
Tellez T, Sanchez Leon Z, Galvao L, Nolder D, Monje Rumi M, Levi JE, Ramirez JD, Zorrilla P, Flores M, Jercic MI, Crisante G, Anez N, De Castro AM, Gonzalez CI, Acosta Viana K, Yachelini P, Torrico F, Robello C, Diosque P, Triana Chavez O, Aznar C, Russomando G, Buscher P, Assal A, Guhl F, Sosa Estani S, DaSilva A, Britto C, Luquetti A, Ladzins J: International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients. PLoS Negl Trop Dis 2011, 5:e931 33. Elnifro EM, Ashshi AM, Cooper RJ, Klapper PE: Multiplex PCR: optimization and application in diagnostic virology. Clin Microbiol Rev 2000, 13:559e570 34. van Doorn R, Klerks MM, van Gent-Pelzer MP, Speksnijder AG, Kowalchuk GA, Schoen CD: Accurate quantification of microorganisms in PCR-inhibiting environmental DNA extracts by a novel internal amplification control approach using Biotrove OpenArrays. Appl Environ Microbiol 2009, 75:7253e7260
144
jmd.amjpathol.org
-
The Journal of Molecular Diagnostics
jmd.amjpathol.org
Multiplex Screening for Blood-Borne Viral, Bacterial, and Protozoan Parasites using an OpenArray Platform Elena Grigorenko,* Carolyn Fisher,y Sunali Patel,* Caren Chancey,y Maria Rios,y Hira L. Nakhasi,y and Robert C. Duncany From Life Technologies Corp.,* South San Francisco, California; and the Division of Emerging and Transfusion Transmitted Diseases,y Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland Accepted for publication August 22, 2013. Address correspondence to Robert C. Duncan, Ph.D., 1401 Rockville Pike, HFM310, Rockville, MD 20852. E-mail: [email protected].
The use of nucleic acid tests for detection of pathogens has improved the safety of blood products. However, ongoing pathogen emergence demonstrates a need for development of devices testing for multiple pathogens simultaneously. One approach combines two proven technologies: Taqman chemistry for target identification and quantification and the OpenArray nanofluidic real-time PCR platform for spatial multiplexing of assays. A panel of Taqman assays was developed to detect nine blood-borne pathogens (BBPs): four viral, two bacterial, and three protozoan parasites. The custom BBP OpenArray plate with 18 assays was tested for specificity and analytical sensitivity for nucleic acid from each purified pathogen and with pathogen-spiked human blood and plasma samples. For most targets, the limits of detection (10 to 10,000 copies/mL) were comparable with existing real-time platforms. The testing of the BBP OpenArray with pathogen-spiked coded human plasma or blood samples and negative control specimens demonstrated no false-positive results among the samples tested and correctly identified pathogens with the lowest concentration detected ranging from 10 cells/mL (Trypanosoma cruzi) to 10,000 cells/mL (Escherichia coli). These results represent a proof of concept that indicated the BBP OpenArray platform in combination with Taqman chemistry may provide a multiplex real-time PCR pathogen detection method that points the way for a next-generation platform for infectious disease testing in blood. (J Mol Diagn 2014, 16: 136e144; http://dx.doi.org/10.1016/j.jmoldx.2013.08.002)
Testing of donations for blood-borne pathogens (BPPs) has reduced significantly the risk of transfusion transmission of such agents. However, the current number of pathogen tests and the increasing number of emerging pathogens that can affect blood safety results in an increased number of screening tests required before release of the blood and blood products. The use of a single pathogen assay for each agent is burdensome and costly and requires volumes of specimens that risk exceeding the maximum collection volume allowed for testing. Devices that allow simultaneous testing for multiple pathogens (multiplex testing) can potentially streamline blood donation testing. To date, the highest number of pathogens that can be assayed in one multiplex test licensed for blood screening is five [HIV-1 group M and group O, HIV-2, hepatitis C virus (HCV), and hepatitis B virus (HBV)].1 Current multiplex tests require subsequent discriminatory assays to identify which pathogen produced the reactive test result. An improved technology using real-time nucleic acid tests Copyright ª 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jmoldx.2013.08.002
(NATs) that promise to deliver discrimination simultaneously with detection is available in Europe but not the United States (TaqScreen MPX Test, version 2.0; Roche, Roche Molecular Diagnostics, Pleasanton, CA) and other devices that combine amplification with microarray hybridization have also been proposed.2 New technology needs to be developed that can achieve in a single assay both higher levels of multiplicity and simultaneous discrimination of the reactive agent. Application of novel and emerging technologies offers the prospect to develop the next generation of superior performance tests that Supported in part by an internal FDA grant under the Modernizing Science Program. Disclosures: E.G. and S.P. are employees of Life Technologies Corp., the manufacturer of the OpenArray platform. The comments in this publication are an informal communication and represent the authors’ own best judgments. These comments do not bind or obligate the FDA. The devices described in this publication have not been formally cleared or approved by the FDA for the uses discussed herein.
Multiplex Blood-Borne Pathogen Screening will detect an increasing number of transfusion-transmittable agents in multiplex format with high sensitivity and specificity, robustness, and adaptability to accomplish detection of new agents. To date, NATs have only been applied to viral pathogens, namely, HIV, HCV, HBV, and West Nile virus (WNV) using plasma specimens. Testing of plasma specimens would be substantially improved if all of these viral NAT assays could be performed on a single device with simultaneous detection and discrimination. Further, no Food and Drug Administrationeapproved NAT is being performed for blood donor screening of whole blood specimens. Nucleic acid detection of bacteria and protozoan parasites, such as Plasmodium, Trypanosoma cruzi, and Leishmania, that would be associated with whole blood specimens would make a significant contribution to blood safety if available on a multiplex assay of sufficient sensitivity. A multiplex approach can be achieved by using assays with different reporters combined in the same reaction well or by using high densities of physically independent PCR wells. However, both approaches have challenges. The presence of multiple primers may lead to cross-hybridization with each other and the possibility of mispriming with other templates, so optimization is required for any new primer sets in multiplex PCR. As for the other approach, stringent fluidic isolation between adjacent PCR reaction wells is necessary to prevent cross-contamination during loading and temperature cycling on any PCR platform with a high density of independent wells. Recent advancements in high-throughput realtime PCR technologies, such as the OpenArray platform (Life Technologies Corp., Carlsbad, CA), address these disadvantages by assembling selected primer and probe sets in spatially isolated reaction wells and combine the sensitivity and specificity of quantitative PCR with the highthroughput performance of microarrays to achieve largescale screening of pathogens.3e5 In this study, we evaluated a custom BBP OpenArray panel composed of primer and probe sets developed by scientists from both our institutions and published sources.6e8 We determined its limit of detection for four viral pathogens in human plasma specimens simultaneously and five bacterial and protozoan pathogens in human blood specimens simultaneously. We also evaluated the custom BBP OpenArray’s ability to correctly identify coded specimens at the limit of detection.
Materials and Methods Pathogen Culture and Specimen Preparation HIV, HCV, and HBV were prepared from Center for Biologics Evaluation and Research (CBER) lot release panel members that were composed of human plasma specimens from infected individuals or infected cell culture, diluted with pooled human plasma defibrinated and delipidated, and tested negative for common BBPs (base matrix) to 500 or 100
The Journal of Molecular Diagnostics
-
jmd.amjpathol.org
copies/mL, inactivated, and stored in 1-mL vials at 80 C. The viral load of the frozen vials was extensively validated, as previously described for HIV9,10 and HCV.11 The HBV panel members were derived from source material that was a specimen obtained from a blood donor in the United States [HBV DNA, genotype A, serotype adw2; Food and Drug Administration (FDA) identification No. 723534]. The panel members were diluted in base matrix and tested to contain 100 and 500 DNA copies/mL (Steve Kerby, FDA, unpublished data). The lot release panel members used were obtained directly from the Product Testing Section, Division of Emerging and Transfusion Transmitted Diseases, CBER, FDA. WNV was cultured on Vero cells,12 and the supernatant was collected and viral RNA quantified by both plaqueforming units per milliliter determined by plaque assay13 and copies per milliliter in limiting dilution and a TaqMan assay.14,15 Two bacterial species, Escherichia coli and Yersinia enterocolitica, were cultured in Super Broth (Quality Biological Inc., Gaithersburg, MD) and quantified by measuring optical density at 600 nm and converting to cells per milliliter by the formula 5.0 108 cells/mL Z 1 absorbance unit with the SmartSpec 3000 spectrophotometer (BioRad Laboratories Inc., Hercules, CA). Leishmania donovani 1S2D promastigote form was cultured as previously described16 and quantified with a Beckman/Coulter particle counter. T. cruzi Columbiana strain parasites were cultured as epimastigotes as previously described17 and quantified with a Beckman/Coulter particle counter. Plasmodium falciparum was cultured as previously described18 and quantified by microscopic determination of the percentage of red blood cells infected and determination of the number of red blood cells per milliliter with a Beckman/ Coulter particle counter, then the Plasmodium cells were calculated by the product of these two numbers.
Spiking For HIV, HCV, and HBV, the 1-mL aliquoted lot release panel members were used directly as test specimens without further dilution. WNV-spiked specimens were prepared (under biosafety level 3 containment) from live WNV cell culture stock at a concentration of 1010 copies/mL as determined by various methods in collaborative studies. An intermediate dilution of 1:1000 containing 107 copies/mL of WNV was prepared in PBS, followed by a second intermediate dilution of 1:100 in human plasma to have 105 copies/mL, which was further subjected to a 10-fold serial dilution in 1-mL aliquots of human plasma to prepare the testing specimens that contained 104, 103, and 102 copies/ mL. E. coli, Y. enterocolitica, L. donovani, T. cruzi, and P. falciparum were similarly diluted and spiked into whole heparinized human blood. The blood and plasma were obtained from deidentified donors to the research program at the Department of Transfusion Medicine, National Institutes of Health, under an approved protocol (principal investigator R.C.D.).
137
Grigorenko et al Table 1
Assays for Plasma and Whole BBPs Found in Public Sources or Available on Request Amplicon
Species
Target gene
Assay name
Accession No.*
Coordinates
Size (bp)
Reference
HIV HIV HIV HCV Leishmania Leishmania T. cruzi T. cruzi
GAG GAG GAG 3’UTR X-tail Minicircle 18S rRNA Minisatellites Minisatellites
HIV-1A HIV-1B HIV-1C HCV-A LEI-1 LEI-2 TCF-1 TCF-2
JN417241.1 JN417241.1 JX973372 EU835523.1 AB678348.1 FR799614.1 AY520036y AY520036y
684-797 677-755 724-794 3-56 10-125 1020493- 1020707 26-190 26-187
113 78 70 53 115 214 164 161
I. Hewlett, FDA, unpublished data I. Hewlett, FDA, unpublished data R.C.D., FDA, unpublished data 6 7 R.C.D., FDA, unpublished data 8 R.C.D., FDA, unpublished data
*Accession numbers are for GenBank (http://www.ncbi.nlm.nih.gov/genbank). y Two assays only differ by probe sequence. bp, base pair.
Nucleic Acid Extraction Nucleic acid from 140 mL of WNV-spiked plasma was extracted using the QIAamp Viral Mini Kit (Qiagen, Valencia, CA) according to the manufacturer protocol and RNA eluted twice with 100 mL of elution buffer each elution into the same tube. Nucleic acid from 1 mL of all other plasma specimens was extracted with the QIAamp MinElute Virus Spin Kit (Qiagen) according to the manufacturer protocol and eluted in 180 mL of elution buffer. DNA from 1-mL whole blood specimens was extracted with the QIAamp DNA Blood Mini Kit (Qiagen) according to the manufacturer protocol and eluted in 180 mL of elution buffer.
Primer Design and Selection The panel for detection of BBPs consisted of primers and Taqman probes for viral, bacterial, and parasitic targets (Tables 1 and 2) designed for this study, published in the literature or commercially available from Life Technologies Corp. A proprietary algorithm was applied for a subset of Taqman assays designed by Life Technologies Corp. This algorithm evaluates a set of optimal assays, considering criteria of melting temperature and nucleotide composition of primerpair combinations. The algorithm selects the assays with highest specificity, based on nucleic acid sequence comparison of assay primers and probes with genomic sequences from other closely related species. Based on sequence comparison, the assay with the highest mismatch score with other Table 2
organisms was chosen, which minimizes the possibility of generation of false-positive results during the testing. Primers and probes designed by the FDA laboratory as part of this study were selected from genomic sequence using MacVector software version 11.1.2 (MacVector, Inc., Cary, NC), avoiding hairpin loops, primer duplexes, and optimizing melting temperatures.
Reverse Transcription Reaction and Preamplification Nucleic acid solution (10 mL) extracted from plasma samples was reverse transcribed using the High Capacity Reverse Transcription Kit (Life Technologies Corp., P/N 4368814) with random hexamer primers or gene-specific primers (pool of all assay primers diluted 1:5 relative to the concentration in the PCR reaction), in a total volume of 20 mL, incubated at 37 C for 2 hours per manufacturer protocol. To increase sensitivity for targets present at low concentration, targetspecific preamplification was performed. A 10-microliter sample of cDNA was mixed with 10 mL of a mixed pool of PCR assay oligonucleotides at 180 nmol/L-50 nmol/L primer pair-probe concentration and 20 mL of Taqman preamplification master mix (Life Technologies Corp., P/N 4384267). The preamplification reaction was performed on an ABI9700 at the following cycling conditions: single cycle (95 C for 10 minutes), 18 cycles (95 C for 15 seconds and 60 C for 1 minute), and one cycle (95 C for 5 minutes). Preamplification products were diluted 1:10 in 0.1 TE buffer and were used on the BBP OpenArray platform.
Commercial Taqman Assays Available from Life Technologies Corp.
Assay name
LT assay No.
Organism
Gene
Target accession No.
HCV-B HBV-A HBV-B WNV-A WNV-B PLA-1 PLA-2 GNEG-1 GNEG-2
Pa03453408_s1 Pa03453405_s1 Pa03453406_s1 Pa04329496_s1 Pa04329497_s1 Pa04329498_s1 Pa04329499_s1 Pa04329500_s1 Pa04329501_s1
HCV HBV HBV WNV WNV Plasmodium species Plasmodium species Gram-negative bacteria Gram-negative bacteria
50 UTR P, S P, X 30 UTR 30 UTR Mitochondrial rRNA Mitochondrial rRNA gapA gapA
AF009606 X04615 X04615 AF202541.1 AF533540.1 GQ355486.1 AY791633.1 X02662.1 X02662.1
138
jmd.amjpathol.org
-
The Journal of Molecular Diagnostics
Multiplex Blood-Borne Pathogen Screening Table 3
BBP OpenArray Results With Virus-Spiked Plasma Specimens Assay 1
Pathogen (copies/mL) HIV (500) HCV (500) HBV (500) WNV (100) WNV (1000) WNV (10,000)
Assay 2
Assay 3
Name
Mean Cq (95% CI)
No. of positive/total through-holes Name
Mean Cq (95% CI)
HIV-1A HCV-A HBV-A WNV-A WNV-A WNV-A
14.3 NA 18.5 21.3 19.2 14.7
24/24 0/24 24/24 24/24 24/24 24/24
17.4 19.2 19.4 NA 25.8 20.0
(0.1) (0.05) (0.06) (0.05) (0.05)
HIV-1B HCV-B HBV-B WNV-B WNV-B WNV-B
No. of positive/ total throughholes Name
(0.1) 12/24 (0.04) 24/24 (0.04) 24/24 0/24 (0.27) 24/24 (0.07) 24/24
Mean Cq (95% CI)
No. of positive/total through-holes
HIV-1C 14.2 (0.2) 5/24
NA, not applicable.
Real-Time PCR on the BBP OpenArray Platform The OpenArray technology is based on a metal plate the size of a microscope slide that has been photolithographically patterned and etched to form a rectilinear array of 3072 throughholes, organized in 48 subarrays with 64 through-holes each. Each through-hole is loaded with individual Taqman assays and contains 33 nL of PCR mixture. Previous work4 has shown that the PCR assay performance in the nanoplates is equivalent to the same assay in microplates but with a >150-fold lower reaction volume (33-nL versus 5-mL PCR reaction volumes) and with the ability to profile multiple targets using the same sample. cDNA and genomic DNA samples were tested on custom BBP OpenArray plates containing 18 Taqman assays spotted in triplicate. Genomic DNA or cDNA (1.2 mL of each) was mixed with 1.3 mL of PCR-grade water and 2.5 mL of Taqman OpenArray real-time PCR master mix (Life Technologies Corp., P/N 4462159). Then, the reaction mixture was dispensed on a BBP OpenArray plate using the automated sample loading system Accufill (Life Technologies, Corp.). Real-time PCR occurred in an OpenArray custom computercontrolled imaging thermal cycler under software control, where imaging data for up to 9216 individual reaction wells (three plates) were collected during 40 cycles of PCR. The Table 4
postacquisition step includes calculation of quantification cycle (Cq) and the CT confidence parameter, which were used for further data analysis and in the decision tree for pathogen identification. The CT confidence parameter is calculated using Life Technologies Corp.’s proprietary algorithm, and its value reflects the quality of the amplification curve generated during PCR. The CT confidence values >300 indicate good quality of amplification. The CT confidence values of 200 through 300 are considered marginal, and each amplification curve with such CT confidence values has to be manually checked before the result can be considered valid. Reactions with CT confidence values 50% Through-holes/assay for one assay/pathogen Yes
Ct Con.30*
No
Reproducibility Some Through-holes positive for all assays/pathogen
No
Negative
Yes Specificity Cq values and Through-hole count substantially higher than all other pathogens
No
Indeterminate
Figure 1 The decision tree for interpretation of BBP OpenArray results. This flow diagram indicates the series of questions that would be asked of each assay result, then the results of each pair of assays that detect a particular pathogen to determine whether the results indicate that pathogen has been detected. The flow initiates at the upper left corner. Ct Con, Ct confidence (a measure of the quality of the amplification curve). Asterisk: One exception to this rule is made for the P. falciparum assays, which have a higher level of specificity; Cq values up to 35 are accepted to indicate the presence of that pathogen.
Yes
Retest
Pathogen Identified
random primers or a pool of gene-specific primers, then preamplified for 18 cycles with a pool of all 18 primer pairs. A 1:10 dilution of this preamplification mix was used as template in the BBP OpenArray. Two criteria were used for assessment of a standard experiment with BBP OpenArray plates: counting the number of through-holes with amplification detected for each reactive assay of the total number of through-holes used for that assay and the mean Cq values for the reactive through-holes. A series of experiments that led to the determination of the limits of detection for each virus from spiked samples are given in Table 3, where the results are reported for the three assays for HIV and two assays for the other viruses. This set of experiments suggested that on a BBP OpenArray plate, HIV and HBV could be detected below 500 copies/mL, HCV was not consistently detected at 500 copies/mL, and WNV could be detected consistently at 1000 copies/mL. This is an unexpectedly high concentration for the limit of detection for WNV and will be discussed further below. Unspiked control specimens (pathogen-free plasma) were also tested on the same OpenArray plates and showed no amplification for any assays. On the basis of comparison of amplification results for each assay, a set of criteria (mean Cq values and the CT confidence parameter, which reflects the quality of the amplification curve, reproducibility among the replicates of
The Journal of Molecular Diagnostics
-
jmd.amjpathol.org
an assay, reproducibility among the assays targeting a particular pathogen, and assay specificity) were compiled that completely distinguished a true-positive result from a true-negative result among the spiked plasma specimens with known virus concentrations or plasma alone. These criteria were expressed in a decision tree (Figure 1) that was applied to all of the replicates of each assay for each sample to reach a decision regarding the presence or absence of each pathogen in each sample. Guided by this decision tree, we proceeded to evaluate blinded specimens. To evaluate the ability of the BBP OpenArray plates to identify unknown viral specimens, plasma was spiked with measured amounts of cultured pathogens or selected CBER lot release panel members (see Materials and Methods) near the limits of detection determined above. The testing of 25 coded nucleic acid specimens, including pathogen-free plasma on BBP OpenArray plates, resulted in 16 truepositive, four true-negative, five false-negative, and no false-positive results (Table 4), which is a success rate of 80% (20/25; 95% CI, 60.9%e91.1%). Among these results were three specimens of WNV at 100 copies/mL and one at 10 copies/mL, all detected successfully, indicating the expected lower limit of detection is achievable for WNV on the BBP OpenArray plates. Most of the false-negative results occurred in the specimens with the lowest virus concentrations (100 copies/mL).
141
Grigorenko et al
Evaluation of Whole Blood Specimens
A BBP multiplex real-time PCR device has been assembled that could screen a blood specimen for four virus species, three protozoan parasite species, and a whole group of bacteria simultaneously by splitting the sample into plasma and red blood cell fractions. The tested pathogens, HBV, HCV, HIV-1, WNV, gram-negative bacteria, Leishmania, Plasmodium, and T. cruzi, are all agents that pose a significant risk for transmission in blood or blood component transfusion. These pathogens were all correctly identified in blinded testing on a BBP OpenArray platform at limits of detection of a slightly higher pathogen concentration than those observed with lower multiplicity tests that detect virus in plasma23e27 or DNA genome pathogens in blood.28e32 This study represents a demonstration of the proof of concept for an advance in multiplexing of blood donor screening and diagnostics. The extraction method using offthe-shelf column-based technology can ensure reproducibility for DNA and RNA yields from clinical specimens. In optimizing the reverse transcription step, gene-specific primers were selected because they led to generally higher cDNA yield than random primers. To achieve lower limits
of detection, an initial preamplification step was added wherein a larger volume of specimen with a pool of the primers with the same sequence as those on the BBP OpenArray platform was thermocycled 10 to 18 times. The addition of a preamplification PCR reaction to improve sensitivity increases the risk of contamination as reported by others.33 However, care in handling, separation of PCR setup and PCR product manipulation into different rooms, and monitoring each reaction with no-template control tubes eliminated false-positive results in our hands. Optimization of the BBP OpenArray benefited from extensive prior experience with microorganism detection with this platform.3,4,34 The flexibility of the BBP OpenArray platform derived from user selection of assays, which can be chosen from user designed primers and probes or from a commercial vendor. Many of the primers used in this study were used previously as an optimized assay in singletube, 384-well or 96-well plates. Some additional primers had been designed specifically for this study and underwent experimental validation for specificity and sensitivity. The choice of primer sequence also involved optimizing amplicon length, a property that must balance efficiency of amplification (shorter length) with specificity of detection (sufficient length to accommodate an optimal fluorescent hydrolysis probe). The chosen layout of the assays, each subarray with the set of 18 assays in triplicate, meant that 48 specimens could be amplified on one BBP OpenArray plate (one specimen to each subarray) with each specimen tested three times with each assay. Alternatively, a specimen could be loaded on multiple subarrays, increasing the number of replicates but decreasing the total number of specimens that could be processed per BBP OpenArray plate. This alternative was our preferred approach because the larger number of replicates enhanced the sensitivity and specificity. The BBP OpenArray performance was validated with whole human blood and plasma spiked with the agent in 1 mL volume at the specified concentration. This approach ensures that all of the inhibitors that might be present in infected human blood or plasma are present and the extraction technique is challenged to recover the pathogen genome from the volume and complexity of a natural sample. Of course, subtle differences remain in an actual infected human specimen, so consequently testing clinically derived samples will be the subject of future investigations. The eightfold level of multiplicity achieved represents a substantial improvement in streamlining blood donor screening and clinical diagnostics. However, this is only proof of the concept that the BBP OpenArray can perform detection with microorganismcontaminated blood and plasma. This platform has capacity to expand to higher levels of multiplicity. The flexibility of the platform is also a much desired asset, allowing addition or removal of assays as new agents emerge or crises, such as bioweapons, are deployed. The existence of a fully validated platform that can adapt with only minor modification is needed. The design of the BBP OpenArray may fulfill this need because each assay is performed in a separate through-
142
jmd.amjpathol.org
The pathogens targeted for detection in whole blood specimens were all DNA genome organisms, such as bacteria and protozoan parasites. Volunteer blood was spiked with cultured pathogens at known concentrations and DNA extracted, and the nucleic acid sample was tested on the BBP OpenArray plates. Multiple runs with specimens spiked at 10-fold dilutions showed reproducible and specific detection at the highest concentration but intermittent success at lower concentrations (Table 5). A preamplification step is widely used for gene expression profiling to increase sensitivity of the detection in real-time PCR when starting material is limited.21,22 To achieve reliable detection of pathogens at the lower concentrations, each whole blood specimen was preamplified with a pool of all of the assay primers and probes before loading on the BBP OpenArray plates. The criteria in the pathogen identification decision tree (Figure 1) developed for the plasma specimens were applied without modification to the assessment of the whole blood specimens. Unknown specimens were prepared by spiking cultured bacteria and protozoan parasites into whole blood at known concentrations near and below the limits of detection determined above. Twenty-three coded specimens were tested at least three times on separate OpenArray plates with 9 to 18 replicate through-holes for each assay. The decision tree was used to make a determination from the combined results of the three runs for each specimen (Table 6). Overall, among the 23 specimens, there were 22 correct calls (true-positive or true-negative results, 96% (95% CI, 79.5%e99.3%) and one incorrect call (4%). There were no false-positive results.
Discussion
-
The Journal of Molecular Diagnostics
Multiplex Blood-Borne Pathogen Screening hole with little likelihood that a new assay would interfere with an existing one. Novel technology has been applied to a recognized problem in testing blood and blood products for infectious agents. The BBP OpenArray platform customized with BBP real-time PCR assays has demonstrated a higher level of multiplicity with sensitivity and specificity, suggesting that the concept can be further developed to achieve suitability for clinical use.
13.
14.
15.
Acknowledgments We thank Jiangqin Zhao, Indira Hewlett, Stephen Kerby, Nancy Lee, Victoria Majam, Sanjai Kumar, Rana Nagarkatti, and Alain Debrabant (all from CBER, FDA, Bethesda, MD) for primer sequences and pathogen source material.
References
16.
17.
18.
1. Margaritis AR, Brown SM, Seed CR, Kiely P, D’Agostino B, Keller AJ: Comparison of two automated nucleic acid testing systems for simultaneous detection of human immunodeficiency virus and hepatitis C virus RNA and hepatitis B virus DNA. Transfusion 2007, 47:1783e1793 2. Tomioka K, Peredelchuk M, Zhu X, Arena R, Volokhov D, Selvapandiyan A, Stabler K, Mellquist-Riemenschneider J, Chizhikov V, Kaplan G, Nakhasi H, Duncan R: A multiplex polymerase chain reaction microarray assay to detect bioterror pathogens in blood. J Mol Diagn 2005, 7:486e494 3. Gonzales TK, Kulow M, Park DJ, Kaspar CW, Anklam KS, Pertzborn KM, Kerrish KD, Ivanek R, Dopfer D: A high-throughput open-array qPCR gene panel to identify, virulotype, and subtype O157 and non-O157 enterohemorrhagic Escherichia coli. Mol Cell Probes 2011, 25:222e230 4. Morrison T, Hurley J, Garcia J, Yoder K, Katz A, Roberts D, Cho J, Kanigan T, Ilyin SE, Horowitz D, Dixon JM, Brenan CJ: Nanoliter high throughput quantitative PCR. Nucleic Acids Res 2006, 34:e123 5. Slinger R, Moldovan I, Barrowman N, Chan F: Successful nanolitre real-time PCR detection of respiratory pathogens in clinical specimens. Clin Microbiol Infect 2012, 18:E286eE288 6. Drexler JF, Kupfer B, Petersen N, Grotto RM, Rodrigues SM, Grywna K, Panning M, Annan A, Silva GF, Douglas J, Koay ES, Smuts H, Netto EM, Simmonds P, Pardini MI, Roth WK, Drosten C: A novel diagnostic target in the hepatitis C virus genome. PLoS Med 2009, 6:e31 7. Nicolas L, Prina E, Lang T, Milon G: Real-time PCR for detection and quantitation of leishmania in mouse tissues. J Clin Microbiol 2002, 40: 1666e1669 8. Piron M, Fisa R, Casamitjana N, Lopez-Chejade P, Puig L, Verges M, Gascon J, Gomez i Prat J, Portus M, Sauleda S: Development of a realtime PCR assay for Trypanosoma cruzi detection in blood samples. Acta Trop 2007, 103:195e200 9. Davis C, Heath A, Best S, Hewlett I, Lelie N, Schuurman R, Holmes H: Calibration of HIV-1 working reagents for nucleic acid amplification techniques against the 1st international standard for HIV-1 RNA. J Virol Methods 2003, 107:37e44 10. Lee S, Wood O, Taffs RE, Hu J, Machuca A, Vallejo A, Hewlett I: Development and evaluation of HIV-1 subtype RNA panels for the standardization of HIV-1 NAT assays. J Virol Methods 2006, 137: 287e291 11. Saldanha J, Heath A, Lelie N, Pisani G, Nubling M, Yu M: Calibration of HCV working reagents for NAT assays against the HCV
The Journal of Molecular Diagnostics
12.
-
jmd.amjpathol.org
19.
20. 21.
22.
23.
24.
25.
26.
27.
28.
international standard: The Collaborative Study Group. Vox Sang 2000, 78:217e224 Grinev A, Daniel S, Laassri M, Chumakov K, Chizhikov V, Rios M: Microarray-based assay for the detection of genetic variations of structural genes of West Nile virus. J Virol Methods 2008, 154:27e40 Rios M, Zhang MJ, Grinev A, Srinivasan K, Daniel S, Wood O, Hewlett IK, Dayton AI: Monocytes-macrophages are a potential target in human infection with West Nile virus through blood transfusion. Transfusion 2006, 46:659e667 Rios M, Daniel S, Chancey C, Hewlett IK, Stramer SL: West Nile virus adheres to human red blood cells in whole blood. Clin Infect Dis 2007, 45:181e186 Rios M, Daniel S, Dayton AI, Wood O, Hewlett IK, Epstein JS, Caglioti S, Stramer SL: In vitro evaluation of the protective role of human antibodies to West Nile virus (WNV) produced during natural WNV infection. J Infect Dis 2008, 198:1300e1308 Debrabant A, Joshi MB, Pimenta PFP, Dwyer DM: Generation of Leishmania donovani axenic amastigotes: their growth and biological characteristics. Intl J Parasitol 2004, 34:205e217 McDaniel JP, Dvorak JA: Identification, isolation, and characterization of naturally-occurring Trypanosoma cruzi variants. Mol Biochem Parasitol 1993, 57:213e222 Mahajan B, Zheng H, Pham PT, Sedegah MY, Majam VF, Akolkar N, Rios M, Ankrah I, Madjitey P, Amoah G, Addison E, Quakyi IA, Kumar S: Polymerase chain reaction-based tests for pan-species and species-specific detection of human Plasmodium parasites. Transfusion 2012, 52:1949e1956 Sokal R, Rohlf F: Biometry: The Principles and Practice of Statistics in Biological Research. ed 2. New York, W. H. Freeman and Company, 1981 Wilson EB: Probable inference, the law of succession, and statistical inference. J Am Stat Assoc 1927, 22:209e212 Li J, Smyth P, Cahill S, Denning K, Flavin R, Aherne S, Pirotta M, Guenther SM, O’Leary JJ, Sheils O: Improved RNA quality and TaqMan Pre-amplification method (PreAmp) to enhance expression analysis from formalin fixed paraffin embedded (FFPE) materials. BMC Biotechnol 2008, 8:10 Noutsias M, Rohde M, Block A, Klippert K, Lettau O, Blunert K, Hummel M, Kuhl U, Lehmkuhl H, Hetzer R, Rauch U, Poller W, Pauschinger M, Schultheiss HP, Volk HD, Kotsch K: Preamplification techniques for real-time RT-PCR analyses of endomyocardial biopsies. BMC Mol Biol 2008, 9:3 Araujo F, Henriques I, Monteiro F, Meireles E, Cruz A, Tavares G, Mota-Miranda A: Evaluation of NucliSens-AmpliScreen methodology to detect subtypes G of HIV-1 and 4c/4d of HCV in the screening of blood donors. Transfus Clin Biol 2005, 12:331e335 Busch MP, Tobler LH, Saldanha J, Caglioti S, Shyamala V, Linnen JM, Gallarda J, Phelps B, Smith RI, Drebot M, Kleinman SH: Analytical and clinical sensitivity of West Nile virus RNA screening and supplemental assays available in 2003. Transfusion 2005, 45: 492e499 Li L, Chen PJ, Chen MH, Chak KF, Lin KS, Tsai SJ: A pilot study for screening blood donors in Taiwan by nucleic acid amplification technology: detecting occult hepatitis B virus infections and closing the serologic window period for hepatitis C virus. Transfusion 2008, 48: 1198e1206 Pichl L, Heitmann A, Herzog P, Oster J, Smets H, Schottstedt V: Magnetic bead technology in viral RNA and DNA extraction from plasma minipools. Transfusion 2005, 45:1106e1110 Pisani G, Pupella S, Marino F, Gaggioli A, Sambri V, Rossini G, Wirz M, Grazzini G: Interlaboratory study to evaluate the performance of laboratories involved in West Nile virus RNA screening of blood and blood components by nucleic acid amplification testing in Italy. Blood Transfus 2011, 9:425e429 Mary C, Faraut F, Lascombe L, Dumon H: Quantification of Leishmania infantum DNA by a real-time PCR assay with high sensitivity. J Clin Microbiol 2004, 42:5249e5255
143
Grigorenko et al 29. Najdenski H, Heyndrickx M, Herman L, Werbrouck H, Van Coillie E: Quantification of Yersinia enterocolitica in raw milk using qPCR. Vet Microbiol 2012, 160:428e434 30. Patel P, Garson JA, Tettmar KI, Ancliff S, McDonald C, Pitt T, Coelho J, Tedder RS: Development of an ethidium monoazideenhanced internally controlled universal 16S rDNA real-time polymerase chain reaction assay for detection of bacterial contamination in platelet concentrates. Transfusion 2012, 52:1423e1432 31. Schachterle SE, Mtove G, Levens JP, Clemens EG, Shi L, Raj A, Munoz B, Reller ME, West S, Dumler JS, Sullivan D: Prevalence and density-related concordance of three diagnostic tests for malaria in a region of Tanzania with hypoendemic malaria. J Clin Microbiol 2011, 49:3885e3891 32. Schijman AG, Bisio M, Orellana L, Sued M, Duffy T, Mejia Jaramillo AM, Cura C, Auter F, Veron V, Qvarnstrom Y, Deborggraeve S, Hijar G, Zulantay I, Lucero RH, Velazquez E,
Tellez T, Sanchez Leon Z, Galvao L, Nolder D, Monje Rumi M, Levi JE, Ramirez JD, Zorrilla P, Flores M, Jercic MI, Crisante G, Anez N, De Castro AM, Gonzalez CI, Acosta Viana K, Yachelini P, Torrico F, Robello C, Diosque P, Triana Chavez O, Aznar C, Russomando G, Buscher P, Assal A, Guhl F, Sosa Estani S, DaSilva A, Britto C, Luquetti A, Ladzins J: International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients. PLoS Negl Trop Dis 2011, 5:e931 33. Elnifro EM, Ashshi AM, Cooper RJ, Klapper PE: Multiplex PCR: optimization and application in diagnostic virology. Clin Microbiol Rev 2000, 13:559e570 34. van Doorn R, Klerks MM, van Gent-Pelzer MP, Speksnijder AG, Kowalchuk GA, Schoen CD: Accurate quantification of microorganisms in PCR-inhibiting environmental DNA extracts by a novel internal amplification control approach using Biotrove OpenArrays. Appl Environ Microbiol 2009, 75:7253e7260
144
jmd.amjpathol.org
-
The Journal of Molecular Diagnostics
