Literature
Aims: CYP2C19 variant alleles are independent predictors of clopidogrel response variability and occurrence of major adverse cardiovascular events in high-risk vascular patients on clopidogrel therapy. Increasing evidence suggests a combination of platelet function testing with CYP2C19 genetic testing may be more effective in identifying high-risk individuals for alternative antiplatelet therapeutic strategies. A crucial point in evaluating the use of these polymorphisms in the clinical practice, besides test accuracy, is the cost of the genetic test and rapid availability of the results. One hundred acute coronary syndrome patients were genotyped for CYP2C19*2,*3,*4,*5, and *17 polymorphisms with two platforms: Verigene® and the TaqMan® system. Results: Genotyping results obtained by the classical TaqMan approach and the rapid Verigene approach showed a 100% concordance for all the five polymorphisms investigated. The Verigene system had shorter turnaround time with respect to the TaqMan. The cost of reagents for TaqMan genotyping was lower than that for the Verigene system, but the effective manual staff involvement and the relative cost resulted in higher cost for TaqMan than for Verigene. Conclusions: The Verigene system demonstrated good performance in terms of turnaround time and cost for the evaluation of the clopidogrel poor metabolizer status, giving genetic information in suitable time (206 min) for a therapeutic strategy decision.
Aiming to improve the performance of tumor marker assays, earlier we have developed a microarray based nano-probe system. Our tumor assay format is broadly applicable across various protein biomarkers and uses a multi-step deterministic robotic process, which relies on non-isotropically oriented antibodies on functionalized glass as multiplexed microarrays to capture tumor markers from serum or plasma. Functionalized gold probes, which are in the order of a few hundred angstroms diameter, also bind to the tumor markers through a molecular-scale complex containing antibody. The tumor marker-bound molecular complex is then quantified through silver enhancement of the functionalized gold.
Based on the ultrasensitive nano-assay system, we have developed a novel assay format for detection of tumor markers for prostate cancer which utilizes the autoantibodies present in patient serum or plasma. This format is coined as ‘nanofishing’ test. This new assay format was characterized and compared to the conventional mode of tumor marker detection. Based on the ultra-sensitive gold nano-particle probe to detect tumor markers, such nanofishing assays could provide a powerful tool for evaluating novel biomarkers, validation and clinical applications, including early diagnosis and prognosis.
In Japan, immunochromatography is the diagnostic test of choice in the diagnosis of influenza virus infection. However, its sensitivity is affected by the time between onset of disease and when testing is performed. On the other hand, PCR has significantly higher sensitivity; however, testing is performed in the laboratory and results are not available on a timely basis to have clinical impact. During the period December 2010 to April 2011, three hospitals affiliated with Juntendo University assessed a bed-side, high sensitivity molecular assay (Verigene RV+, Nanosphere, USA) against immunochromatography with respect to impact on patient management. A total of 103 pediatric patients with symptoms suggesting influenza infection were tested by both immunochromatography (Poctem InfluenzaAB, Sysmex Corporation, Japan) and Verigene RV+ (RV+). Our results show that the RV+ sensitivity was significantly higher than immunochromography; furthermore, the most significant difference was seen during the 3 hour period between early onset of fever and testing. This finding has implications for infection control in the pediatric population during triaging at admissions. Furthermore, the timely availability of a high sensitive molecular assay suggests that the RV+ assay will have a positive impact on outpatient pediatric patient management
Numerous drugs such as clopidogrel have been developed to reduce coagulation or inhibit platelet function. The hepatic cytochrome P450 (CYP) pathway is involved in the conversion of clopidogrel to its active metabolite. A recent black-box warning was included in the clopidogrel package insert indicating a significant clinical link between specific CYP2C19 genetic variants and poor metabolism of clopidogrel. Of these variants, (*)2 and (*)3 are the most common and are associated with complete loss of enzyme activity. In patients who are carriers of a CYP2C19 (*)2 or (*)3 allele, the conversion of clopidogrel to its active metabolite may be reduced, which can lead to ischemic events and negative consequence for the patient. We examined the ability of the Verigene CLO assay (Nanosphere, Northbrook, IL) to identify CYP2C19 (*)2 and (*)3 polymorphisms in 1,286 unique whole blood samples. The Verigene CLO assay accurately identified homozygous and heterozygous (*)2 and (*)3 phenotypes with a specificity of 100% and a final call rate of 99.7%. The assay is fully automated and can produce a result in approximately 3.5 hours.
Background: Bacteremia and sepsis result in high morbidity and mortality. A majority of positive blood cultures are due to gram‐ positive bacteria including Staphyloccocus, Enterococcus, Streptococcus, and Micrococcus. Some of these organisms are associated with serious infection, while others are skin flora associated with lines or improperly collected specimens. Early identification of the organism and appropriate antibiotic treatment is critical to management of the infection and improving patient outcome.
Methods: Sixty positive blood cultures containing gram‐positive bacteria were analyzed using the Verigene Gram Positive Blood Culture (BC‐GP) Assay (Nanosphere, Northbrook, IL). Testing of cultures was done within 8h of positivity. The BC‐GP detects 13 bacterial targets including Staphylococcus, Streptococcus, Enterococcus, Micrococcus and Listeria species. The resistance determinants mecA (oxacillin) and vanA/B (vancomycin) are also detected.
Results: Three cultures contained organisms not on the BC‐GP panel (Bacillus, Lactobacillus), and were resulted as “Not Detected”. Compared to biochemical identification, 55 of 57 positive blood cultures were concordant with BC‐GP results. One culture contained viridans group Streptococci by biochemical identification, which was discordant with the BC‐GP result (S. pneumoniae). In 4 of 5 cultures containing two organisms the BC‐GP correctly identified both organisms; in one culture the BC‐GP identified 1 of 2 organisms present. This translates to a combined sensitivity of 98.4%. The BC‐GP predicted oxacillin resistance in 34 Staphylococcus spp. (18 mecA +) and vancomycin resistance in 10 Enterococcus spp. (6 vanA +) for 100% sensitivity and specificity.
Conclusions: The BC‐GP assay identifies 13 gram‐positive targets and 2 resistance markers directly from positive blood cultures. The BC‐GP requires only 350μl of specimen and results are available within 2.5 hours of blood culture positivity.
INTRODUCTION
Timely and sensitive diagnosis of influenzavirus (IF) and respiratory syncytial virus (RSV) infections in hospitalised patients is important for infection control and clinical management. PCR testing offers greater sensitivity that traditional methods, but methodologies for rapid and flexible automated testing have only recently become available. We performed a beta test evaluation of the Verigene Respiratory Virus Plus nucleic acid test (RV+, Nanosphere Inc.), which detects and types RSV and IF, including detection of oseltamivir resistance in seasonal influenza H1N1 and influenza H1N1 (2009).
METHODS
Clinical specimens submitted to HPA Microbiology Services Bristol for investigation of suspected respiratory virus infection were tested for IF and RSV by real-time PCR (1,2). Samples testing positive for influenza H1N1 (2009) were further tested for genotypic evidence of oseltamivir resistance using a real-time PCR to discriminatea major single nucleotide polymorphism encoding a point mutation the neuraminidase gene (H275Y) which confers oseltamivir resistance(3). Most of these samples were also tested by pyrosequencing analysis of N1 sequences to determine the proportions of sensitive and resistant virus (4). A panel of samples testing positive or negative for these viruses was selected from samples with sufficient residual volume after completion of routine diagnostic investigations for evaluation of RV+. Fully automated nucleic acid extraction, multiplex reverse transcription PCR amplification and microarray hybridization was performed using a Verigene Processor SP(Figure 1a). Microarrays were then transferred to a Verigene Reader to analyse and report results (Figure 1b). Results of RV+ testing were then compared with those of the comparator laboratory tests.
RESULTS
The RV+ assay was found to be technically straightforward, and delivered unequivocal results in 2.5 hours with approximately 5 minutes hands-on time per sample. The results of the detection of IF and RSV types by RV+ in samples previously characterised by real-time PCR for these viruses is summarised in Table 1. Further details of results of genotypic detection of oseltamivir resistance in IFA H1N1 2009 positive samples by RV+, real-time PCR and pyrosequencing are shown in Table 2 (discordant results shaded magenta).
DISCUSSION
We have compared the RV+ assay with highly sensitive and specific molecular diagnostic assays, and not with less sensitive virus culture or antigen detection methods, as is common in commercial test evaluations. Nevertheless, the RV+ showed good concordance with results of the comparator test. RV+ did not detect or report oseltamivir resistance in 3/21 samples in which resistant virus was detected by pyrosequencing; a mixture of sensitive and resistant virus was detected in two of these. Samples with <50% resistant virus detected by pyrosequencing are not officially reported as oseltamivir resistant. The diagnostic and prognostic significance of detecting minor populations of resistant virus requires further investigation.
CONCLUSIONS
Background and aims: We evaluated a new modular near-patient PCR system in 2 centres between January and March 2011
Methods: 146 children with fever and respiratory symptoms presenting to the emergency service at Coimbra(C) had nasopharyngeal samples and clinical data collected prospectively with consent. 67 samples collected routinely at Bristol(B) during the same period were studied. At each centre, sample sets were analysed using the Verigene modular PCR system for Influenza A(H1, H1 2009,H3),B, RSV A&B & Oseltamivir resistance mutation H275Y. The B set was also analysed using established PCR assays.
Results: By Verigene, in C, 15,0&48/146 were positive for InflA,B,RSV(24A,24B) (4 double positives(dp)) and 87 were negative. The corresponding B results were 27,22&8(5A,3B)/67 (2dp) &12 negative. In C-B 14-25 InflA were H1 2009, 0-1 H3, 1-1 untyped (none H275Y+ in C, 14/25 & 2 indeterminate in B). Results by established assays in B were concordant except the untyped H1 sample which was positive for H1 2009, the indeterminate samples which were H275Y+ & 2 H275Y negatives which were weakly positive. In C, 6/15 InflA+ ( 1 received Oseltamivir) and 16/48 RSV+ children were admitted.
Conclusions: The Verigene results matched those from established assays closely. The small number of discrepant results generally related to weakly reactive or indeterminate samples. Evident clinical benefit of rapid-near patient testing included RSV bed-management and cohorting decisions and guidance of Oseltamivir use in seriously sick children. The system would also permit tracking of local outbreak epidemiology in centres without ready access to viral PCR testing
Introduction: Multiplexed molecular detection of influenza A, influenza B, and respiratory syncytial virus (RSV) is becoming increasingly available with a number of commercial assays on the market. However, there are no ‘sample-to-result’ platforms available that detect these 3 respiratory virus types along with influenza A and RSV subtypes (H1, H3, H1N12009) and (A and B), respectively. Numerous studies have demonstrated increased sensitivity of nucleic acid-based techniques compared to culture and antigen-based detection methods. This study evaluated the sensitivity and specificity of the Verigene Respiratory Virus Plus Nucleic Acid Test (RV+) (Nanosphere, Northbrook, IL) for the detection of these respiratory viruses and subtypes.
Methods: A total of 1022 nasopharyngeal specimens were collected prospectively at three geographically distinct sites during the 2008-9 and 2009-10 respiratory seasons. The presence of influenza A/B and RSV was determined at the time of collection using culture/DFA techniques. Subtyping for influenza A and RSV was accomplished using bidirectional sequencing. Residual frozen samples were divided among 3 test sites which conducted testing on each specimen using the RV+. The culture/DFA and bidirectional sequencing results were blinded to the study investigators until the conclusion of the study. Discrepant results were resolved using bi-directional sequencing.
Results: Across all 3 test sites, 988 of 1022 specimens generated a definitive result after initial analysis (initial call rate 96.7%). All but 2 of these samples generated a definitive result upon retest (final call rate 99.8%). Of the 1022 specimens enrolled in the clinical trial, 315 (30.8%) were culture/DFA positive for influenza A, 43 (4.2%) were positive for influenza B, 107 (10.5%) were positive for RSV. In comparison to culture/DFA for influenza A, the RV+ assay was 98.7% sensitive and 93.2% specific. Upon resolution of 52 discrepant results, the RV+ sensitivity and specificity were 100%. Notably, this included the detection of an additional 44 influenza A positive specimens missed by culture/DFA. For influenza B, the RV+ assay was 100% sensitive and 99.7% specific. Specificity was 100% following discrepant resolution. For RSV, the RV+ assay was 99.1% sensitive and 100% specific. Of the 354 sequence confirmed influenza A specimens, 207 were subtyped as 2009 H1N1. The RV+ assay identified 206 of the 207 2009 H1N1 specimens, indicating 99.5% sensitivity and 100% specificity for this subtype. For the H1 (n=39) and H3 (n=108) subtypes, the sensitivity and specificity was ≥ 99.9%. The RV+ assay was also 100% sensitive and specific for identifying RSV A (n=57) and B (n=53) subtypes.
Conclusion: The RV+ assay for detection and subtyping of respiratory viruses offers a rapid result time of ~2.5 hours with sensitivity and specificity equal to or greater than traditional culture-based detection methods. Additionally, the fully automated platform allows simple, on demand assay set up which permits workflow flexibility and requires minimal hands on time.
Introduction: The Verigene® Respiratory Virus Plus Nucleic Acid Test (RV+) is an FDA cleared sample-to-answer, nucleic acid amplification, array hybridization, nanoparticle probe-based assay for the detection of influenza and RSV. The RV+ assay detects and differentiates influenza A, influenza A subtypes (H1, H3, and 2009 H1N1), influenza B, and RSV subtypes (A and B). The purpose of this study was to determine the range of analytical reactivity of the RV+ assay inclusive of influenza H5 and H7 subtypes.
Methods: For this analytical reactivity study, three (3) H5N1 and two (2) H7N7 influenza A viral subtypes were prepared as follows. Viruses were grown, titered, heat inactivated, and confirmed sterile prior to use. Qualification of the heat inactivated virus stocks was performed using real-time RT-PCR on an ABI 7500®utilizing primers and probes specific for the Influenza A M2 gene. Additionally, subtypes were confirmed using primers and probes specific for H5 and H7 gene markers. Relative titers (TCID50/mL) of the inactivated virus were calculated based on the differences in the Ct values between RNA amplified from live virus prior to inactivation and the subsequent inactivated virus stocks. A known positive Influenza A clinical sample was included in each qualification run both as a positive control and as a benchmark for typical clinical titer. Upon qualification, dilutions of 1:1000, 1:10,000 and 1:100,000 for each inactivated virus subtype were made fresh prior to each run. Two hundred microliters of each dilution was run in triplicate on the RV+ test.
Results: Heat inactivation had a slight effect on relative virus titer as detected at the nucleic acid level. On average, there was a 1 log loss (approximately 3 Ct) in relative titer due to the inactivation of the 5 virus subtypes. The qualified inactivated viruses were tested in three (3) concentrations with relative titers ranging from 5.3 E0 to 2.8 E6 (TCID50). The H5 and H7 strains gave a ‘Detected’ result for Influenza A and a ‘Not Detected’ result for H1, H3, and 2009 H1N1. As expected, these strains were unsubtypeable in the RV+ assay. No cross-reactivity with H1, H3, or 2009 H1N1 was observed at any of the dilutions, including higher concentrations, further demonstrating good assay specificity. In addition, all extracts produced from the RV+ assay were confirmed as either H5 or H7 and relative virus titers confirmed by real-time RT-PCR.
Conclusion: All dilutions of the H5 and H7 heat inactivated virus samples produced positive results and appropriate dose responses in the RV+ assay. Based on these results, the Nanosphere Verigene® RV+ test has the ability to detect a wide range of influenza A subtypes with high sensitivity and specificity.
Introduction: Multiplexed molecular detection of influenza A and B and RSV is becoming increasingly available with a number of commercial assays on the market. However, there are no ‘sample-to-result’ platforms that detect these viruses along with influenza A and RSV subtypes.
Numerous studies have demonstrated increased sensitivity of these techniques compared to culture and antigen detection methods. This study evaluated the sensitivity and specificity of Nanosphere’s Verigene System, with the Processor SP, for the detection of these respiratory
viruses.
Methods: Nasopharyngeal specimens were collected at multiple sites during the 2008−9 and 2009−10 respiratory seasons. The presence of influenza A/B and RSV was determined using culture/DFA. Influenza A and RSV subtypes were determined using bi-directional sequencing. Discrepant results were resolved using bi-directional sequencing. The culture/DFA and bi-directional sequencing results for the samples were blinded to the investigators until the conclusion of the study.
Results: Of the 323 specimens enrolled in the clinical trial, 121 were culture/DFA positive for Influenza A, 14 were positive for influenza B, 33 were positive for RSV, and 155 specimens were culture/DFA negative. In comparison to culture/DFA for influenza A, the RV+ assay was 100% sensitive and 97.0% specific. For influenza B, the RV+ assay was 100% sensitive and 100% specific. For RSV, the RV+ assay was 97.0% sensitive and 99.3% specific. There were 9 discrepant specimens between RV+ and culture/DFA. Bidirectional sequencing revealed that the RV+ assay resulted in 8 additional true positives which were otherwise negative by culture/DFA, and 1 additional true negative which was resulted as positive by culture/DFA. All positive influenza A samples were subtyped as 2009 H1N1 by sequencing (n = 127). The RV+ assay performance for this subtype was 99.2% sensitivity and 100% specificity. For the H1 and H3 subtypes, the specificities ranged from 99.7–100%. For the RSV A and B subtypes, the sensitivities and specificities were 100% and 100%, respectively, for both subtypes.
Conclusion: The RV+ assay for detection and subtyping of respiratory viruses offers a rapid result time of ~2.5 hours with sensitivity and specificity equal to or greater than traditional culture-based detection methods. Additionally, the fully automated platform allows simple, on demand assay set-up which permits workflow flexibility and requires minimal hands on time.