- Email: [email protected]
Automation of laboratory testing for infectious diseases using the polymerase chain reaction — our past, our present, our future
Journal of Clinical Virology 20 (2001) 1 – 6 www.elsevier.com/locate/jcv
Review
Automation of laboratory testing for infectious diseases using the polymerase chain reaction — our past, our present, our future Donald Jungkind * Clinical Microbiology Laboratories, Department of Pathology, Thomas Jefferson Uni6ersity, Philadelphia, PA 19107, USA
Abstract While it is an extremely powerful and versatile assay method, polymerase chain reaction (PCR) can be a labor-intensive process. Since the advent of commercial test kits from Roche and the semi-automated microwell Amplicor™ system, PCR has become an increasingly useful and widespread clinical tool. However, more widespread acceptance of molecular testing will depend upon automation that allows molecular assays to enter the routine clinical laboratory. The forces driving the need for automated PCR are the requirements for diagnosis and treatment of chronic viral diseases, economic pressures to develop more automated and less expensive test procedures similar to those in the clinical chemistry laboratories, and a shortage in many areas of qualified laboratory personnel trained in the types of manual procedures used in past decades. The automated Roche COBAS AMPLICOR™ system has automated the amplification and detection process. Specimen preparation remains the most labor-intensive part of the PCR testing process, accounting for the majority of the hands-on-time in most of the assays. A new automated specimen preparation system, the COBAS AmpliPrep™, was evaluated. The system automatically releases the target nucleic acid, captures the target with specific oligonucleotide probes, which become attached to magnetic beads via a biotin–streptavidin binding reaction. Once attached to the beads, the target is purified and concentrated automatically. Results of 298 qualitative and 57 quantitative samples representing a wide range of virus concentrations analyzed after the COBAS AmpliPrep™ and manual specimen preparation methods, showed that there was no significant difference in qualitative or quantitative hepatitis C virus (HCV) assay performance, respectively. The AmpliPrep™ instrument decreased the time required to prepare serum or plasma samples for HCV PCR to under 1 min per sample. This was a decrease of 76% compared to the manual specimen preparation method. Systems that can analyze more samples with higher throughput and that can answer more questions about the nature of the microbes that we can presently only detect and quantitate will be needed in the future. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Polymerase chain reaction; Automation; AmpliPrep; Routine diagnostic laboratory
* Corresponding author. Tel.: + 1-215-9558726; fax: + 1-215-9236039. E-mail address: [email protected] (D. Jungkind). 1386-6532/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 6 - 6 5 3 2 ( 0 0 ) 0 0 1 4 8 - 7
2
D. Jungkind / Journal of Clinical Virology 20 (2001) 1–6
1. Introduction Polymerase chain reaction (PCR) is an extremely powerful and versatile assay method, but it can be a labor-intensive process when using the manual home-brew methods (Klapper et al., 1998). Since the advent of commercial test kits from Roche and the semi-automated microwell AMPLICOR™ system, PCR has become an increasingly useful and widespread clinical tool (Bass et al., 1993; Beavis et al., 1995). In our experience, most persons in the clinical laboratory workforce are not well trained in molecular biology methods. Widespread use of routine PCR testing in clinical laboratories depends upon automation (Jungkind 1996; Klapper et al., 1998; Lisby, 1999). There are at least three forces driving the need for automated PCR. The first is the new standards of care requiring molecular based viral load tests for treatment of chronic viral diseases. The second is the economic pressures on clinical laboratories to produce more work with disproportionate increases in labor, space, and funding. This has accentuated the need to develop more automated and less expensive test procedures similar to those in the clinical chemistry laboratories. Finally, there is emerging in some areas a shortage of qualified laboratory personnel trained in both the classical and molecular microbiological procedures. The classical microbiology laboratory requires natural products such as tissue cultures, sheep red blood cells, and extracts of plant and animal products for growth of microbes. Animals or cell cultures for production of antibodies are used in immunological assays. New methods of detection and identification of microbes will be increasingly needed in years to come as man begins longer explorations of space, and considers manned stations on the moon and Mars. Man will certainly take his infections along wherever he goes in this new millennium. For journeys to Mars that may be measured in years, classical microbiology techniques would not be easy to maintain, and the advantages of an automated chemistry style microbiology test would become apparent. While that is still in the future, the other considerations mentioned create an immediate need to begin this technological journey
to automate the clinical microbiology laboratory. Clinical microbiology is at the stage of the clinical chemistry laboratories of the 1960s when classical manual chemistry methods were just beginning to be replaced with automated instrumentation. Managing the direction and pace of this development will be an exciting challenge for microbiologists and clinicians in years to come. The first automated PCR amplification and detection instrument for the clinical laboratory was the Roche COBAS AMPLICOR™ system (Jungkind et al., 1996; Kessler et al., 1999; Van der Pol et al., 2000). This greatly decreased the amount of hands-on time needed for producing a PCR result. It took approximately 20–60 min of hands-on time to produce a PCR result using early manual PCR methods if one includes a portion of the time required for initial test validation and routine maintenance of properly working reagents (Klapper et al., 1998). The hands-on time for the first commercial kit PCR test (Chlamydia trachomatis) was approximately 3 min to produce a test result using the Roche microwell assay. For the COBAS AMPLICOR™ system, the first automated amplification and detection PCR instrument, the hands-on time was 3.3 min to produce three PCR results (Chlamydia trachomatis, Neisseria gonorrhoeae, and internal control results; Jungkind et al., 1996). The COBAS Amplicor™ system took 7.8 min of hands-on time to produce a PCR result for hepatitis C virus (HCV), because of the longer time for the specimen preparation step (Klapper et al., 1998). With the viral assays, specimen preparation is critical to test accuracy, yet it remained the most labor-intensive part of the PCR testing process, accounting for the majority of the hands-on-time (Verhofstede et al., 1996; Deggerdal and Larsen, 1997; Klapper et al., 1998). The increasing importance of viral load assays in the management of patients with chronic virus infections has increased the need for easier procedures to accomplish this new laboratory task (Garson et al., 1990; Bagasra et al., 1998; Afonso et al., 2000; Pawlotsky et al., 2000). A new automated specimen preparation system, the COBAS AmpliPrep™, was developed to automate this last part of the PCR procedure. The system automatically releases the target nucleic acid, captures the
D. Jungkind / Journal of Clinical Virology 20 (2001) 1–6
target with specific oligonucleotide probes, which become attached to magnetic beads via a biotin – streptavidin binding reaction. Once attached to the beads, the target is purified and concentrated automatically by the instrument.
2. Automated specimen preparation studies Research studies with the AmpliPrep™ instrument were started in the Clinical Microbiology Laboratories, Department of Pathology, Thomas Jefferson University in April 1998. All specimen preparation procedures were performed on samples as described in the package insert of the Roche COBAS AMPLICOR™ HCV quantitative Monitor assay. HCV positive and negative serum and plasma samples were supplied by Roche Molecular Systems, Pleasanton, CA. Samples were obtained from sites in the USA and Europe and were stored frozen at − 70°C for most of the time, and briefly at −20°C during transport. For the correlation studies between manual and AmpliPrep™ HCV PCR results, a pre-prototype instrument (functional module) was used. It was identical in function to later instruments except it lacked some of the convenience, contamination control, throughput enhancements, and reliability features that were later included on the prototype instrument. Our initial study was a determination of the linearity of the results of known titer diluted samples. Linearity was determined using known HCV viral titers prepared by diluting a HCV standard and preparing the dilution samples using the AmpliPrep™ instrument. The prepared samples were analyzed six times using the Roche COBAS Amplicor™ HCV Monitor assay and the average titer of each sample was determined. The linearity correlation was determined using a plot comparing the log of the standard titer vs the averaged AmpliPrep™ titer for each data point. In regression curve comparison of the AmpliPrep™ versus standard test results, the R 2 was 0.99. Qualitative and quantitative correlation studies were performed using the COBAS AMPLICOR™ HCV Monitor kits supplied by Roche Molecular
3
Systems. The specimen preparation was performed using manual methods as described in the package insert and by the automated AmpliPrep™ system. A total of 355 samples were analyzed after AmpliPrep™ and manual specimen preparation. Of the 298 qualitative samples, 180 positive samples and 94 negative samples gave concordant results. There were 14 AmpliPrep™ negative and manual test positive samples, and ten AmpliPrep™ positive and manual test negative samples. Of the 24 instances of discrepancy, 16 were due to specimens with less than 2000 HCV copies/ ml. The detection limit of the HCV assay at the time of these studies was 2000 HCV copies/ml. Therefore, two-thirds of our discrepant samples represented random positives from samples below the threshold of the assay. Six of the remaining eight discrepant samples were less than 5000 HCV copies/ml, and only two samples gave values higher than 10 000. Of those two samples, one was missed by the AmpliPrep™ method and the other was missed by the manual method. There was equivalence between the two methods of specimen preparation at the qualitative level. Fifty-seven quantitative samples were analyzed using the COBAS AMPLICOR™ Monitor assay after both the COBAS AmpliPrep™ method and the manual specimen preparation method. The results of these comparisons showed that the virus levels were comparable. In regression curve comparison of the AmpliPrep™ and manual test results, the R 2 of the 57 quantitative samples was 0.91. We used the prototype instrument for all tests for cross contamination between samples, endurance runs, and workflow studies. AmpliLink™ software was available for the prototype model of the AmpliPrep™ unit. This made that instrument more convenient to use. For possible cross contamination studies, four negative controls in each of 36 runs were interspersed with positive HCV control samples during processing on the prototype model of the AmpliPrep™ instrument. These were subsequently tested with the COBAS AMPLICOR™ Monitor assay. All 144 negative samples remained negative after processing and analysis. Likewise, 31 nega-
4
D. Jungkind / Journal of Clinical Virology 20 (2001) 1–6
tive specimens interspersed with positive specimens were also processed by the manual specimen preparation method, and all 31 tested negative with the COBAS AMPLICOR™ HCV Monitor assay. The contamination rate was estimated to be below 0.7%, but because no false positives were detected, we do not know how low the final rate will be. Further studies are needed to determine the exact number. Endurance runs were made by running 3504 samples through the AmpliPrep™ instrument over several weeks and recording the percentage of successful sample preparations and the percentage of samples that had to be restarted in order to complete the processing task. In these tests of reliability of the prototype instrument, we found that 95.7% of the specimens were successfully processed on the first attempt. Most of the 4.3% that failed only had to have the run restarted and were completed on the same day. The rest were delayed at least a day to repair or adjust the instrument. By extending the endurance runs, we were able to identify several areas for improvement in the reliability of the instrument. These changes have already been made in the next version of the instrument. Further changes will be made after evaluating the second prototype, before production instruments will be available. Other convenience factors were the availability of ready to use reagents, and the ability to track specimens using barcodes, to insure a higher degree of operator success. Specimen stability after preparation on the AmpliPrep™ instrument indicated that specimens could be left on the instrument for 5 days at room temperature with no significant change in titer. Completed specimen preparations could also be removed from the instrument and stored at 4°C for 5 days with no significant change in titer. The effect of anticoagulants on the virus levels was determined by collecting a sample from a patient with a titer of approximately 4× 105 HCV copies/ml. Otherwise identical specimens were collected in a plain glass blood collection tube, and in standard blood collection tubes containing EDTA, heparin, ACD, or sodium citrate. Virus levels in the samples were determined using the COBAS Amplicor™ Monitor HCV assay after
both manual and AmpliPrep™ specimen preparation. There was no significant difference in the virus titer between any of these sample anticoagulant types. There were no significant differences between the manual and COBAS Ampliprep™ virus levels. The timing study techniques originally used by the College of American Pathologists in their workload timing studies were used to study the COBAS AmpliPrep™ process. Waiting time while the instrument was processing specimens was not included in the calculation of hands-on time unless the operator was doing tasks related to the specimen preparation process during the time that the instrument was running. If the technologist had no more work to do related to the specimen preparation procedure, it was assumed that he would be able to perform other testing duties in the laboratory. Averages were made by timing two technologists to produce the hands-on time required for the manual vs the AmpliPrep™ specimen preparation methods. The AmpliPrep™ instrument decreased the time required to prepare serum or plasma samples for HCV PCR to just under 1 min per sample. This was a decrease of 76% compared to the manual specimen preparation method, which took just over 4 min per sample. Using the continuous run mode of the AmpliPrep™ instrument, we could easily process 144 samples in 8.4 h with only 2.3 h of actual handson time. The continuous run mode was most efficient, because it allowed us to continue to reload the instrument with specimens and reagents while it was processing specimens. When running 144 samples in non-continuous batch mode (two batches of 72 samples) one must wait to finish one batch before starting the next. In batch mode, the total time to completion of all 144 samples was extended to 10.3 h. The Roche AmpliPrep™ instrument completes the automation of the final area in the PCR process. The specimens must be placed on the COBAS AmpliPrep™ instrument and the processed specimens have to be added to the master mix reagents in the ring of tubes for the COBAS AMPLICOR™ instrument. Future refinements should allow further integration of the successors
D. Jungkind / Journal of Clinical Virology 20 (2001) 1–6
to these two instruments so that the specimen would only have to be handled once by the technologist.
3. Conclusions This represents the first report of total automation of all steps in PCR using a commercially available clinical testing system. Further advances will improve the functionality of first generation automation, but this automation is a significant advance over previous systems, and makes it possible for automated PCR to enter many laboratories that would otherwise be unable to provide this service due to personnel or space restrictions. The COBAS AmpliPrep™ system replaced a clean room for specimen preparation. With this example of total automation of a clinical microbiology process to detect and quantify an infectious agent, nucleic acid amplification methods may provide the way for converting significant amounts of conventional microbiological testing to a chemistry-based assay technique (White et al., 1992; Lisby, 1999). If clinical microbiology follows the path of clinical chemistry, new and better instrumentation will emerge and the testing menu will be expanded. Where competition between various nucleic acid technologies can produce similar results, improvements will follow and prices may fall (Goessens et al., 1997). Molecular testing has been most successful in areas for which conventional microbiological techniques do not exist, are too slow, or are too expensive. Virology has benefited greatly from these procedures. For widespread use in other areas of the clinical microbiology laboratory, there must be major technological breakthroughs in addition to the gradual evolutionary changes that will follow the current instruments. One major capability, which must be achieved before automated molecular methods can begin to replace the routine bacterial culture and sensitivity test is the ability to quickly and inexpensively analyze many genetic markers to determine genus, species, and susceptibility type. This may involve analysis of genomic, plasmid, and m-RNA. Because of these unknowns, traditional bacteriology testing will be
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needed for some time to come. Basic research efforts must continue to map and characterize the genes involved, interactions of these genes, and gene expression, to reproduce the information provided by the conventional culture and sensitivity test. During this process new and unexpected correlations may emerge that may change our direction and the way we use laboratory tests as an aid in diagnosis and treatment of infections. Host interactions with the infecting organism are important to the outcome of the infection, but presently we don’t understand these interactions well enough to create the first test in that area. We must be ready to adapt to new information and to adopt these new methods where possible. Careful clinical trials are necessary to validate each new step forward, but we are in the beginning stages of an exciting new era for clinical microbiology.
Acknowledgements This project was supported in part by grants from Roche Molecular Systems.
References Afonso AM, Didier J, Plouvier E, Falissard B, Ferey MP, Bogard M, Dussaix E. Performance of an automated system for quantification of hepatitis C virus RNA. J Virol Methods 2000;86:55 – 60. Bagasra O, Steiner RM, Ballas SK, Castro O, Dornadula G, Embury S, Jungkind D, Bobroski L, Kutlar A, Burchott S. Viral burden and disease progression in HIV-1-infected patients with sickle cell anemia. Am J Hematol 1998;59:199 – 207. Bass CA, Jungkind DL, Silverman NS, Bondi JM. Clinical evaluation of a new polymerase chain reaction assay for detection of Chlamydia trachomatis in endocervical specimens. J Clin Microbiol 1993;31:2648 – 53. Beavis KG, Lichty MB, Jungkind DL, Giger O. Evaluation of Amplicor PCR for direct detection of Mycobacterium tuberculosis from sputum specimens. J Clin Microbiol 1995;33:2582 – 6. Deggerdal A, Larsen F. Rapid isolation of PCR-ready DNA from blood, bone marrow, and cultured cells, based on paramagnetic beads. Biotechniques 1997;22:554 – 7. Garson JA, Tedder RS, Briggs M, Tuke P, Glazebrook JA, Trute A, Parker D, Barbara J, Contreras M, Aloysius S.
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Detection of viral hepatitis C sequences in blood donations by ‘nested’ polymerase chain reaction and prediction of infectivity. Lancet 1990;335:1419–22. Goessens WH, Mouton JW, van der Meijden WI, Deelen S, van Rijsoort-Vos TH, Lemmens-den Toom N, Verbrugh HA, Verkooyen RP. Comparison of three commercially available amplification assays, AMP CT, LCx, and COBAS AMPLICOR, for detection of Chlamydia trachomatis in first-void urine. J Clin Microbiol 1997;35:2628 – 33. Jungkind DL. Evaluation of an automated COBAS AMPLICOR PCR system for detection of Chlamydia trachomatis/Neisseria gonorrhoeae and the impact on laboratory management. In: Stary A, editor. Proceedings of the Third Meeting of the European Society for Chlamydia Research. European Society for Chlamydia Research and Austrian Society for Dermatology and Venerology, 1996:272 – 3. Jungkind DL, DiRenzo S, Beavis KG, Silverman NS. Evaluation of an automated COBAS AMPLICOR PCR system for detection of several infectious agents and the impact on laboratory management. J Clin Microbiol 1996;34:2778– 83. Kessler HH, Jungkind D, Stelzl E, Direnzo S, Vellimedu SK, Pierer K, Santner B, Marth E. Evaluation of AM-
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PLILINK software for the COBAS AMPLICOR system. J Clin Microbiol 1999;37:436 – 7. Klapper P, Jungkind D, Fenner T, Antinozzi R, Schirm J, Blanckmeister C. Multicenter international work flow study of an automated polymerase chain reaction instrument. Clin Chem 1998;44:1737 – 9. Lisby G. Application of nucleic acid amplification in clinical microbiology. Molecular Biotechnology 1999;12:75 – 99. Pawlotsky JM, Bastie A, Hezode C, Lonjon I, Darthuy F, Remire J, Dhumeaux D. Routine detection and quantification of hepatitis B virus DNA in clinical laboratories: performance of three commercial assays. J Virol Methods 2000;85:11 – 21. Van der Pol B, Quinn TC, Gaydos CA, Crotchfelt K, Schachter J, Moncada J, Jungkind D, Martin DH, Turner B, Peyton C, Jones RB. Multicenter evaluation of the AMPLICOR and automated COBAS AMPLICOR CT/ NG tests for detection of Chlamydia trachomatis. J Clin Microbiol 2000;38:1105 – 12. Verhofstede C, Fransen K, Marissens D, Verhelst R, van der Groen G, Lauwers S, Zissis G, Plum J. Isolation of HIV-1 RNA from plasma: evaluation of eight different extraction methods. J Virol Methods 1996;60:155 – 9. White TJ, Madej R, Persing DH. The polymerase chain reaction: clinical applications. Adv Clin Chem 1992;29:161 – 96.
Review
Automation of laboratory testing for infectious diseases using the polymerase chain reaction — our past, our present, our future Donald Jungkind * Clinical Microbiology Laboratories, Department of Pathology, Thomas Jefferson Uni6ersity, Philadelphia, PA 19107, USA
Abstract While it is an extremely powerful and versatile assay method, polymerase chain reaction (PCR) can be a labor-intensive process. Since the advent of commercial test kits from Roche and the semi-automated microwell Amplicor™ system, PCR has become an increasingly useful and widespread clinical tool. However, more widespread acceptance of molecular testing will depend upon automation that allows molecular assays to enter the routine clinical laboratory. The forces driving the need for automated PCR are the requirements for diagnosis and treatment of chronic viral diseases, economic pressures to develop more automated and less expensive test procedures similar to those in the clinical chemistry laboratories, and a shortage in many areas of qualified laboratory personnel trained in the types of manual procedures used in past decades. The automated Roche COBAS AMPLICOR™ system has automated the amplification and detection process. Specimen preparation remains the most labor-intensive part of the PCR testing process, accounting for the majority of the hands-on-time in most of the assays. A new automated specimen preparation system, the COBAS AmpliPrep™, was evaluated. The system automatically releases the target nucleic acid, captures the target with specific oligonucleotide probes, which become attached to magnetic beads via a biotin–streptavidin binding reaction. Once attached to the beads, the target is purified and concentrated automatically. Results of 298 qualitative and 57 quantitative samples representing a wide range of virus concentrations analyzed after the COBAS AmpliPrep™ and manual specimen preparation methods, showed that there was no significant difference in qualitative or quantitative hepatitis C virus (HCV) assay performance, respectively. The AmpliPrep™ instrument decreased the time required to prepare serum or plasma samples for HCV PCR to under 1 min per sample. This was a decrease of 76% compared to the manual specimen preparation method. Systems that can analyze more samples with higher throughput and that can answer more questions about the nature of the microbes that we can presently only detect and quantitate will be needed in the future. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Polymerase chain reaction; Automation; AmpliPrep; Routine diagnostic laboratory
* Corresponding author. Tel.: + 1-215-9558726; fax: + 1-215-9236039. E-mail address: [email protected] (D. Jungkind). 1386-6532/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 6 - 6 5 3 2 ( 0 0 ) 0 0 1 4 8 - 7
2
D. Jungkind / Journal of Clinical Virology 20 (2001) 1–6
1. Introduction Polymerase chain reaction (PCR) is an extremely powerful and versatile assay method, but it can be a labor-intensive process when using the manual home-brew methods (Klapper et al., 1998). Since the advent of commercial test kits from Roche and the semi-automated microwell AMPLICOR™ system, PCR has become an increasingly useful and widespread clinical tool (Bass et al., 1993; Beavis et al., 1995). In our experience, most persons in the clinical laboratory workforce are not well trained in molecular biology methods. Widespread use of routine PCR testing in clinical laboratories depends upon automation (Jungkind 1996; Klapper et al., 1998; Lisby, 1999). There are at least three forces driving the need for automated PCR. The first is the new standards of care requiring molecular based viral load tests for treatment of chronic viral diseases. The second is the economic pressures on clinical laboratories to produce more work with disproportionate increases in labor, space, and funding. This has accentuated the need to develop more automated and less expensive test procedures similar to those in the clinical chemistry laboratories. Finally, there is emerging in some areas a shortage of qualified laboratory personnel trained in both the classical and molecular microbiological procedures. The classical microbiology laboratory requires natural products such as tissue cultures, sheep red blood cells, and extracts of plant and animal products for growth of microbes. Animals or cell cultures for production of antibodies are used in immunological assays. New methods of detection and identification of microbes will be increasingly needed in years to come as man begins longer explorations of space, and considers manned stations on the moon and Mars. Man will certainly take his infections along wherever he goes in this new millennium. For journeys to Mars that may be measured in years, classical microbiology techniques would not be easy to maintain, and the advantages of an automated chemistry style microbiology test would become apparent. While that is still in the future, the other considerations mentioned create an immediate need to begin this technological journey
to automate the clinical microbiology laboratory. Clinical microbiology is at the stage of the clinical chemistry laboratories of the 1960s when classical manual chemistry methods were just beginning to be replaced with automated instrumentation. Managing the direction and pace of this development will be an exciting challenge for microbiologists and clinicians in years to come. The first automated PCR amplification and detection instrument for the clinical laboratory was the Roche COBAS AMPLICOR™ system (Jungkind et al., 1996; Kessler et al., 1999; Van der Pol et al., 2000). This greatly decreased the amount of hands-on time needed for producing a PCR result. It took approximately 20–60 min of hands-on time to produce a PCR result using early manual PCR methods if one includes a portion of the time required for initial test validation and routine maintenance of properly working reagents (Klapper et al., 1998). The hands-on time for the first commercial kit PCR test (Chlamydia trachomatis) was approximately 3 min to produce a test result using the Roche microwell assay. For the COBAS AMPLICOR™ system, the first automated amplification and detection PCR instrument, the hands-on time was 3.3 min to produce three PCR results (Chlamydia trachomatis, Neisseria gonorrhoeae, and internal control results; Jungkind et al., 1996). The COBAS Amplicor™ system took 7.8 min of hands-on time to produce a PCR result for hepatitis C virus (HCV), because of the longer time for the specimen preparation step (Klapper et al., 1998). With the viral assays, specimen preparation is critical to test accuracy, yet it remained the most labor-intensive part of the PCR testing process, accounting for the majority of the hands-on-time (Verhofstede et al., 1996; Deggerdal and Larsen, 1997; Klapper et al., 1998). The increasing importance of viral load assays in the management of patients with chronic virus infections has increased the need for easier procedures to accomplish this new laboratory task (Garson et al., 1990; Bagasra et al., 1998; Afonso et al., 2000; Pawlotsky et al., 2000). A new automated specimen preparation system, the COBAS AmpliPrep™, was developed to automate this last part of the PCR procedure. The system automatically releases the target nucleic acid, captures the
D. Jungkind / Journal of Clinical Virology 20 (2001) 1–6
target with specific oligonucleotide probes, which become attached to magnetic beads via a biotin – streptavidin binding reaction. Once attached to the beads, the target is purified and concentrated automatically by the instrument.
2. Automated specimen preparation studies Research studies with the AmpliPrep™ instrument were started in the Clinical Microbiology Laboratories, Department of Pathology, Thomas Jefferson University in April 1998. All specimen preparation procedures were performed on samples as described in the package insert of the Roche COBAS AMPLICOR™ HCV quantitative Monitor assay. HCV positive and negative serum and plasma samples were supplied by Roche Molecular Systems, Pleasanton, CA. Samples were obtained from sites in the USA and Europe and were stored frozen at − 70°C for most of the time, and briefly at −20°C during transport. For the correlation studies between manual and AmpliPrep™ HCV PCR results, a pre-prototype instrument (functional module) was used. It was identical in function to later instruments except it lacked some of the convenience, contamination control, throughput enhancements, and reliability features that were later included on the prototype instrument. Our initial study was a determination of the linearity of the results of known titer diluted samples. Linearity was determined using known HCV viral titers prepared by diluting a HCV standard and preparing the dilution samples using the AmpliPrep™ instrument. The prepared samples were analyzed six times using the Roche COBAS Amplicor™ HCV Monitor assay and the average titer of each sample was determined. The linearity correlation was determined using a plot comparing the log of the standard titer vs the averaged AmpliPrep™ titer for each data point. In regression curve comparison of the AmpliPrep™ versus standard test results, the R 2 was 0.99. Qualitative and quantitative correlation studies were performed using the COBAS AMPLICOR™ HCV Monitor kits supplied by Roche Molecular
3
Systems. The specimen preparation was performed using manual methods as described in the package insert and by the automated AmpliPrep™ system. A total of 355 samples were analyzed after AmpliPrep™ and manual specimen preparation. Of the 298 qualitative samples, 180 positive samples and 94 negative samples gave concordant results. There were 14 AmpliPrep™ negative and manual test positive samples, and ten AmpliPrep™ positive and manual test negative samples. Of the 24 instances of discrepancy, 16 were due to specimens with less than 2000 HCV copies/ ml. The detection limit of the HCV assay at the time of these studies was 2000 HCV copies/ml. Therefore, two-thirds of our discrepant samples represented random positives from samples below the threshold of the assay. Six of the remaining eight discrepant samples were less than 5000 HCV copies/ml, and only two samples gave values higher than 10 000. Of those two samples, one was missed by the AmpliPrep™ method and the other was missed by the manual method. There was equivalence between the two methods of specimen preparation at the qualitative level. Fifty-seven quantitative samples were analyzed using the COBAS AMPLICOR™ Monitor assay after both the COBAS AmpliPrep™ method and the manual specimen preparation method. The results of these comparisons showed that the virus levels were comparable. In regression curve comparison of the AmpliPrep™ and manual test results, the R 2 of the 57 quantitative samples was 0.91. We used the prototype instrument for all tests for cross contamination between samples, endurance runs, and workflow studies. AmpliLink™ software was available for the prototype model of the AmpliPrep™ unit. This made that instrument more convenient to use. For possible cross contamination studies, four negative controls in each of 36 runs were interspersed with positive HCV control samples during processing on the prototype model of the AmpliPrep™ instrument. These were subsequently tested with the COBAS AMPLICOR™ Monitor assay. All 144 negative samples remained negative after processing and analysis. Likewise, 31 nega-
4
D. Jungkind / Journal of Clinical Virology 20 (2001) 1–6
tive specimens interspersed with positive specimens were also processed by the manual specimen preparation method, and all 31 tested negative with the COBAS AMPLICOR™ HCV Monitor assay. The contamination rate was estimated to be below 0.7%, but because no false positives were detected, we do not know how low the final rate will be. Further studies are needed to determine the exact number. Endurance runs were made by running 3504 samples through the AmpliPrep™ instrument over several weeks and recording the percentage of successful sample preparations and the percentage of samples that had to be restarted in order to complete the processing task. In these tests of reliability of the prototype instrument, we found that 95.7% of the specimens were successfully processed on the first attempt. Most of the 4.3% that failed only had to have the run restarted and were completed on the same day. The rest were delayed at least a day to repair or adjust the instrument. By extending the endurance runs, we were able to identify several areas for improvement in the reliability of the instrument. These changes have already been made in the next version of the instrument. Further changes will be made after evaluating the second prototype, before production instruments will be available. Other convenience factors were the availability of ready to use reagents, and the ability to track specimens using barcodes, to insure a higher degree of operator success. Specimen stability after preparation on the AmpliPrep™ instrument indicated that specimens could be left on the instrument for 5 days at room temperature with no significant change in titer. Completed specimen preparations could also be removed from the instrument and stored at 4°C for 5 days with no significant change in titer. The effect of anticoagulants on the virus levels was determined by collecting a sample from a patient with a titer of approximately 4× 105 HCV copies/ml. Otherwise identical specimens were collected in a plain glass blood collection tube, and in standard blood collection tubes containing EDTA, heparin, ACD, or sodium citrate. Virus levels in the samples were determined using the COBAS Amplicor™ Monitor HCV assay after
both manual and AmpliPrep™ specimen preparation. There was no significant difference in the virus titer between any of these sample anticoagulant types. There were no significant differences between the manual and COBAS Ampliprep™ virus levels. The timing study techniques originally used by the College of American Pathologists in their workload timing studies were used to study the COBAS AmpliPrep™ process. Waiting time while the instrument was processing specimens was not included in the calculation of hands-on time unless the operator was doing tasks related to the specimen preparation process during the time that the instrument was running. If the technologist had no more work to do related to the specimen preparation procedure, it was assumed that he would be able to perform other testing duties in the laboratory. Averages were made by timing two technologists to produce the hands-on time required for the manual vs the AmpliPrep™ specimen preparation methods. The AmpliPrep™ instrument decreased the time required to prepare serum or plasma samples for HCV PCR to just under 1 min per sample. This was a decrease of 76% compared to the manual specimen preparation method, which took just over 4 min per sample. Using the continuous run mode of the AmpliPrep™ instrument, we could easily process 144 samples in 8.4 h with only 2.3 h of actual handson time. The continuous run mode was most efficient, because it allowed us to continue to reload the instrument with specimens and reagents while it was processing specimens. When running 144 samples in non-continuous batch mode (two batches of 72 samples) one must wait to finish one batch before starting the next. In batch mode, the total time to completion of all 144 samples was extended to 10.3 h. The Roche AmpliPrep™ instrument completes the automation of the final area in the PCR process. The specimens must be placed on the COBAS AmpliPrep™ instrument and the processed specimens have to be added to the master mix reagents in the ring of tubes for the COBAS AMPLICOR™ instrument. Future refinements should allow further integration of the successors
D. Jungkind / Journal of Clinical Virology 20 (2001) 1–6
to these two instruments so that the specimen would only have to be handled once by the technologist.
3. Conclusions This represents the first report of total automation of all steps in PCR using a commercially available clinical testing system. Further advances will improve the functionality of first generation automation, but this automation is a significant advance over previous systems, and makes it possible for automated PCR to enter many laboratories that would otherwise be unable to provide this service due to personnel or space restrictions. The COBAS AmpliPrep™ system replaced a clean room for specimen preparation. With this example of total automation of a clinical microbiology process to detect and quantify an infectious agent, nucleic acid amplification methods may provide the way for converting significant amounts of conventional microbiological testing to a chemistry-based assay technique (White et al., 1992; Lisby, 1999). If clinical microbiology follows the path of clinical chemistry, new and better instrumentation will emerge and the testing menu will be expanded. Where competition between various nucleic acid technologies can produce similar results, improvements will follow and prices may fall (Goessens et al., 1997). Molecular testing has been most successful in areas for which conventional microbiological techniques do not exist, are too slow, or are too expensive. Virology has benefited greatly from these procedures. For widespread use in other areas of the clinical microbiology laboratory, there must be major technological breakthroughs in addition to the gradual evolutionary changes that will follow the current instruments. One major capability, which must be achieved before automated molecular methods can begin to replace the routine bacterial culture and sensitivity test is the ability to quickly and inexpensively analyze many genetic markers to determine genus, species, and susceptibility type. This may involve analysis of genomic, plasmid, and m-RNA. Because of these unknowns, traditional bacteriology testing will be
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needed for some time to come. Basic research efforts must continue to map and characterize the genes involved, interactions of these genes, and gene expression, to reproduce the information provided by the conventional culture and sensitivity test. During this process new and unexpected correlations may emerge that may change our direction and the way we use laboratory tests as an aid in diagnosis and treatment of infections. Host interactions with the infecting organism are important to the outcome of the infection, but presently we don’t understand these interactions well enough to create the first test in that area. We must be ready to adapt to new information and to adopt these new methods where possible. Careful clinical trials are necessary to validate each new step forward, but we are in the beginning stages of an exciting new era for clinical microbiology.
Acknowledgements This project was supported in part by grants from Roche Molecular Systems.
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