key: cord-0008428-oy1gbfzh
authors: Hess, Ralf D.; Gärtner, Barbara C.; Garrett, Patricia E.
title: Meeting Report: Part I. Notes from the Molecular Virology Workshop 23–24 April, 2004, Clearwater Beach, Florida, USA
date: 2004-10-18
journal: J Clin Virol
DOI: 10.1016/j.jcv.2004.08.012
sha: 7208de88a4e002e8323cac5dd5797f8c2beba3ef
doc_id: 8428
cord_uid: oy1gbfzh

nan

Frederick S. Nolte (FN), Ph.D., from the Emory University School of Medicine, Atlanta, GA, spoke on Creating a Molecular Diagnostic Laboratory, covering every aspect from space and workflow, to staffing, instruments, costs, reimbursement and regulatory issues. Within the last 5 years, the Molecular Diagnostics Laboratory at Emory experienced a growth in test volume from 9000 tests in 1999 to almost 30,000 tests in 2003. The increased demand for new markers and the availability of open analytical systems has motivated laboratories to develop in-house assays, since the diagnostics industry has not kept pace.

Human Papilloma Virus (HPV) DNA test implementation was a disaster for them. The Cytology Department chose the method they would use, and predicted that they would refer 1000 specimens/year. They actually only referred 500 samples/year in the first few years: lab cost based on a biweekly batch of 20 was $40/test, Medicare reimbursement was $27/test, and turn around time was too long. After 2 years, they gave up and started sending the test out. Some people indicated in the question period that they believe HPV DNA will replace PAP (Papanicoleau smear stain) eventually as a screen for cervical cancer.

Testing for BK Virus (BKV) DNA in renal transplant patients with allograft failure was a big success. They are not only providing good service and getting reimbursed, but the process of organizing everyone who provides care for end stage renal disease (ESRD) to talk about BKV also got everyone talking about multiple other issues and resulted in improved diagnosis and management of nephropathy. (Authors: it does not hurt that ESRD has essentially carte blanche for reimbursement in the U.S.)

From the discussion period • The sample of choice for BKV is blood: there is plenty of signal in urine, but contamination is a problem, and clinical significance is lacking: 30% of transplant patients will have virus in urine, 15% will have viremia, 8% will develop nephropathy. • FN was asked to share knowledge on primers and probes, and offered to share his slides. • For enterovirus he suggested on demand testing, especially during summer time. • Real-time PCR methods provide more precisely quantitative results than traditional PCR, according to one commenter. • Uni-directional workflow is still indicated, but it's no longer so strictly necessary to isolate pre-and post-amplification activities if closed-tube, real-time PCR methods are being exclusively used. • When justifying cost, you have to look at more than costs of reagents and labor: does bringing in the service shorten length of stay, or otherwise save hospital costs? • QC is needed in lot changes for master mix and extraction reagents. • FN's lab runs every test daily, six days a week: he thinks it should be seven. • One questioner brought up the idea of using a single internal control like dolphin HSV 1 for all tests.

In a setting where many labs, probably more than in any other area of clinical diagnostics, are using in-house methods, the next three talks focused on the design, validation and regulation of these methods.

David R. Hillyard, M.D., (DH) from ARUP Laboratories in Salt Lake City, UT spoke on Selecting and Designing Primers and Probes, and his somewhat surprising suggestion was 'don't do it.' At least do not do it from scratch or by yourself. There are many problems with target selection (lack of data to show taxonomic relations and polyphormisms) and sequence design (such as for single nucleotide polymorphism (SNPs)), where very limited published sequence data are available): these will trip up inexperienced investigators. The approach he advised is to order multiple primer sets and use published assays, taking advantage of existing technical and clinical validations, and even then, not to assume that the design works. In the discussion that followed, no one spoke up for de novo design of primers and probes, and several people described problems that arose with new in-house tests: priming failures due to inaccurate melting temperature (T m ) calculations, primer dimer (3'complementarity) and hairpin (self-complementarity) formation, non-specific priming and 3'homology to amplicon. In summary, DH considered SNPs difficult to detect by sequencing, and sequence data bases to be inadequate to identify relevant SNPs for primers and probes.

Alexandra Valsamakis, M.D., (AV) from Johns Hopkins Hospital and Health System, Baltimore, MD described her approach to Validation/Verification of Home-Brew Molecular Assays. She has adapted protocols from various published guidelines (such as NCCLS and College of American Pathologists (CAP)) for the evaluation of accuracy, reproducibility, sensitivity, specificity, limit of detection, and reference or reportable range for qualitative and quantitative tests. Continuing post-verification processes (for about 6 months after implementation) are needed for further data evaluation and gap filling. Mandatory inclusion of external controls and standards from various commercially available sources (e.g., WHO standards for hepatitis A, B, C virus, Human Immunodeficiency Virus (HIV) and parvovirus B19) is recommended. Statistics on LOD (limit of detection) could be done with PROBIT analysis. There are still as yet unidentified inhibitors in polymerase chain reaction (PCR), in addition to known inhibitors such as methanol, heparin, SDS, glycerol and various polysaccharides. AV promoted pragmatism and careful organization as key factors for consistent and useful validation efforts and warned against "winning the battle, but losing the war".

Analyte-Specific Reagents (ASRs): Issues and Challenges was the topic addressed by Sally Hojvat, Ph.D., from the U.S. Food and Drug Administration, Rockville, MD. ASRs are defined in FDA regulations as: "antibodies, both polyclonal and monoclonal, receptor proteins, ligands, nucleic acid sequences, and similar reagents which, through specific binding or chemical reaction with substances in a specimen are intended for use in a diagnostic application for identification and quantification of an individual chemical substance or ligand in biological specimen" (21 CFR, 864, 4020). She described the FDA's intention in promulgating the ASR rule in 1997 to bring the 'active ingredient' in various in-house methods under regulation so that there would be improved oversight of these tests. Under the ASR rule, manufacturers have responsibility for registration and listing of the reagents, manufacture using quality system regulations (QSRs), post-market reporting of serious adverse events, and labelling, according to FDA requirements, with a disclaimer and no clinical or performance claims, and no instructions for use or intended use. ASRs may be sold only to high complexity laboratories. Laboratories have responsibility to function as high complexity labs under the CLIA regulations, to establish performance of tests using ASRs assembled in the laboratory, and to label results with a specific disclaimer: "This test was developed and its performance characteristics determined by (laboratory name). It has not been cleared or approved by FDA." Challenges to the rule have included difficulties with the FDA's ASR database, and the creativity of some companies in marketing devices as ASRs that closely resemble test kits. RUO (research use only) and IUO (investigational use only) diagnostics, in addition to ASR diagnostics, need to be differentiated from FDA-cleared diagnostics. Going forward, FDA will continue to consider how the ASR rule can be better applied to encourage new IVD technologies and diminish concerns about safety and effectiveness, while ensuring quality. Disease Control and Prevention) published case definitions for SARS. While serology is not useful for case definitions (only for epidemiology), either virus culture, which is slow, insensitive and requires level three laboratories, or (better) RT-PCR (reverse transcriptase PCR) from nasopharyngeal swabs, throat swabs, stool, sputum or bronchoscopy specimens should be used. Surprisingly, stool proved to be the best specimen, since it is positive early (6 days after clinical presentation) and still later positive with high titers! In summary, laboratories have a choice in selecting a SARS test: either conventional serology, or real time, inhouse or commercially available RT-PCR assays, based on needs and available instrumentation. There are four commercially available SARS-CoV tests on the market provided by Artus Biotech, Roche, Prodesse and EraGen Biosciences. Three of these had excellent analytical sensitivities using purified RNA, but poor clinical sensitivities from single specimens. Thus, JM's recommendations include testing multiple specimens for SARS-CoV RNA (respiratory plus stool plus follow up specimens), and using a second amplification target as a confirmatory test. Improvement in PCR assays will be required to detect all cases in a timely fashion, and also improvements in sensitivity and specificity of antibody tests will be required for earlier diagnosis. While initial testing by RT-PCR was positive only in 43% (47/109) of the Toronto patients (mean of 3.9 days from onset to collection), convalescent serology was positive in 90% (35/39, mean time of 26 days). RT-PCR was most often positive for specimens collected 9-11 days into the disease.

HMPV, a member of the Paramyxoviridae family classified in the genus Metapneumovirus and subfamily Pneumovirinae, was first isolated in 2001 in the Netherlands. Infections usually occur during the winter months and are common in children. Infections most often affect the lower respiratory tract (similar to RSV, respiratory syncytial virus). The diagnosis of HMPV can be done on LLC-MK2 cells (cytopathic effect is observed as a focal rounding) or by conventional real time PCR assays targeting different genes (nucleocapsid N1, fusion gene F1). Primers for N1 to amplify a 377 bp fragment were suggested as HMPVN1f (forward): 5 -gagaagagctgggtagaag-3 , HMPVN1r (reverse): 5 -gtcttcctgtgctaactttg-3 and for F1 to amplify a 450 bp fragment were HMPVF1f: 5 -ctttggacttaatgacagatg-3 and for HMPVF1r: gtgcttcctgtgctaactttg-3 .

In summary, SARS-CoV and HMPV diagnosis require multiple specimens from multiple sites with multiple techniques. infection are known, depending on the immune status of the patient. EBV seems to be the most successful virus, since more than 90% of elderly are infected. Quantitative assays for EBV viral load (qEBV) are required to diagnose severity of the infection. qEBV may be useful:

1. to predict risk of EBV disease in post-transplant patients, 2. to monitor disease progression, 3. to monitor effectiveness of interventions, and 4. to diagnose primary EBV infections, especially in children.

HB presented data from a series of acute infectious mononucleosis trials among University of Minnesota students. One trial included 19 students (12 had primary infectious mononucleosis as defined on the following serological basis: viral capsid antigen (VCA) IgM positive, VCA-IgG negative and Epstein-Barr nuclear antigen 1 (EBNA-1)-IgG negative) [Authors wonder why also not positive for VCA-IgG, since 'isolated VCA-IgM' is not a common marker in EBV-acute phase sera? Gärtner et al., 2003; Hess, 2004] . For these 12 subjects, the median severity of illness score on a scale of 0 to 6 was 4.0 at day 10 after clinical presentation, which coincided with the highest median concentration of EBV in whole blood (1.5 × 10 4 copies/mL) and peripheral blood monocytes (PBMCs) (4.8 × 10 3 copies/mL). EBV was not commonly found in the plasma; only 16/111 (14%) of the plasma samples tested contained detectable virus. Viral decay in PBMCs paralleled that in whole blood, with a median decay halflife of 4.0 days. The highest concentration of EBV from all sites sampled was in oral washes 6-7 weeks after onset of illness (median 3.5 × 10 5 copies/mL). Five of these subjects had a follow up visit one year after their acute illness, and one still had detectable virus in either the oral pellet or supernatant fluid. In summary, young adults with primary infectious mononucleosis have 10 2 -10 3 more copies of EBV in oral washes, blood and PBMCs than patients with no or past EBV infections.

HB commented on two additional studies on qEBV in oral washes attempting to pinpoint when oral shedding begins to decline and when exactly VCA and EBNA-1 IgG antibodies develop in eight students (7/8 with confirmed infectious mononucleosis). Data are still pending.

(Authors: as evidenced by the discussion that followed, it is far from clear what specimen should be used for viral load, whether cell-free specimens such as plasma or serum, or leucocytes. Also currently we do not know what viral load distinguishes infection from disease, whether or not qEBV can be used to monitor anti-EBV therapy, and whether immune response data can be combined with viral load to predict clinical outcome.)

William Carman, Ph.D., West of Scotland Specialist Virology Centre, Glasgow, Scotland spoke on Molecular Diagnosis of Respiratory Viruses: Is it Necessary? Currently respiratory viruses such as influenza A and B, RSV, parainfluenza 1-4, metapneumoviruses, adenoviruses, enteroviruses, rhinoviruses and others are diagnosed either through direct immunofluorescence (DIF), electron microscopy (EM) or by culture and serology. Comparing results from virus culture to nucleic acid tests (NAT), the NAT or real-time PCR provided the better sensitivities, with clear advantages for testing, e.g., for rhinovirus (<20% in culture versus 80% in NAT), metapneumovirus (<10% culture versus >50% NAT), SARS-CoV (<50% culture versus 80% NAT) or other coronaviruses (<20% culture versus approximately 90% NAT). Culture usually takes several days to complete, while NAT is done within 1-2 days. The positive predictive value (PPV) of NAT may be 90%, while PPV for culture is 100%. Comparing costs of tests, including work load, the DIF was $54/test, culture was $388/test and PCR was $50/test. Thus, the future in respiratory diagnosis was suggested:

1. the end of culture in all but large reference labs, 2. increased knowledge base of clinical implications, 3. improved strategies for adequate epidemiology, 4. potentially, more quantitation of viral load, 5. narrowing the usage of diverse primer/probes, or commercialization of test methods, and 6. lower costs with increased competition. In Vignette #1, there was a good correlation between pleocytosis and EV PCR in CSF, e.g., in patients >2 months of age with no pleocytosis, 98% of patients will be negative on EV RT-PCR (negative predictive value = 98%). In Vignette #2: the major issue in CMV genotyping might be to detect resistance to gancyclovir (GCV), either through culture and plaque assay, PCR/REN (restriction enzyme assay), or through sequencing. In his study only 3% of GCV-resistant viruses had mutations only in the DNA polymerase gene: thus one must use the plaque reduction assay or sequence the UL54 gene (which will also detect mutations that confer cidovir and foscarnet resistance). In Vignette #3: in the early 90's, three nested sets of primers (pol, gag, env) were developed for detecting HIV proviral DNA. All specimens were tested with the pol primer set, and if negative, reported; if positive, confirmed with either gag or env. Now there are re-optimized primers available, e.g., Stratagene Opti-Prime TM kit, providing huge improvements in the gel readout. In Vignette #4: while RSV infections are more seasonal during winter time, B. pertussis infections mainly occur in the summer. Thus, MA recommended not testing for Bordetella pertussis dur-ing the winter respiratory season, on the basis of epidemiological data presented. In Vignette #5: MA's lab tested skin and mucous membrane lesions for HSV with PCR from 1735 specimens, and tested 127 specimens for VZV. Thirty-eight percent (662 samples) were positive for HSV, while 62% were negative (1073). Forty-five percent (57 samples) were positive for VZV and 55% (70) were negative. They did a retrospective study to determine how often VZV DNA was present in lesion specimens where HSV PCR had been ordered (skin and mucous membrane lesions due to HSV and VZV look similar). Results: none of the mucous membrane specimens negative for HSV with PCR, either genital or oral, was VZV PCR positive (0/118 genital specimens; 0/62 oral specimens). VZV DNA was detected in 6 out of 26 (23%) dermal specimens for which VZV PCR was not requested (from HSV PCR negative specimens). He concluded that all dermal lesion specimens submitted for HSV by PCR should be considered for VZV testing, since HSV and VZV are difficult to distinguish visually.

Evaluation of four commercially available Epstein-Barr virus enzyme immuno assays with an immunofluorescence assay as the reference method

Routine Epstein-Barr virus diagnostics from the laboratory perspective: still challenging after 35 years

Hess HiSS Diagnostics GmbH, Colombistrasse 27 79098 Freiburg

Gärtner Department of Virology, Bldg. 47 University of Saarland Medical School D-66421 Homburg/Saar