Performance characterization of the IRIDICA™ Viral IC Assay* for detection and identification of associated with disseminated viral infection in plasma specimens Darcie Baynes, David Metzgar, Megan Rounds, Heather E. Carolan, Donna M. Toleno, David J. Ecker, Rangarajan Sampath, Lawrence B. Blyn Ibis Biosciences, an Abbott Company, Carlsbad, CA, USA

Objectives: Disseminated viral infections commonly affect Results: The LOD of the Viral IC Assay* for Table 3: Potentially cross-reacting organisms. Viruses or Interfering substances study. Table 5b: Carryover study final results. microorganisms that may be present in plasma specimens, or that In order to evaluate the impact of potentially interfering substances on Total number Number of negative samples Carryover Number of valid transplant and chemotherapy recipients, HIV patients, and all tested viruses was ≤ 3500 copies per ml may present similar clinical symptoms to assay targets, were the IRIDICA Viral IC assay’s ability to produce correct assay results, the of carryover detected positive for an rate negative samples other immunocompromised groups. Current standard of in plasma (Table 1). The assay was able to evaluated to determine if they produce potentially cross-reactive 13 assay targets were tested in the presence of 60 potentially interfering opportunities adjacent spiked organism care methods for detecting and identifying the responsible detect and identify all tested strain products that may produce false positive results on the IRIDICA substances. The 56 exogenous substances selected for this study include 70 117* 0 0 Viral IC Assay. The organisms listed below were tested at targeted antiviral, antibiotic, antifungal, and other drugs likely to be * Two samples were excluded from the analysis; one invalid sample resulted in the viruses are targeted towards one or a few viral groups. In variants (Table 2). No interference from medically relevant concentrations of either 1 x 105 units/ml for administered to an immunocompromised patient population (Table 4a, exclusion of one potential carryover event, the other in two potential carryover events. order to provide an inclusive method of identifying drugs or endogenous interfering viruses or 1 x 106 units/ml for bacteria (where units = copies, top). The 4 endogenous substances selected for this study include TCID50, PFU, or CFU, as provided). Each organism was diluted in substances commonly found in plasma specimens (Table 4a, bottom). potentially causal viruses, whether alone or in coinfections, substances was observed, nor was any IRIDICA Negative Control instead of the assay sample type (human Due to their physical nature, these substances could interfere with the a broad-spectrum molecular assay based on mismatch- carryover or cross-reactivity to untargeted plasma). Human plasma is at risk of containing genuine targets of assay’s ability to extract, amplify, or detect viral nucleic acids found in Table 6: Assay reproducibility at 3x LOD. Assay reproducibility was tolerant multiplex PCR and electrospray ionization mass organisms or viruses (Tables 3-5). The the IRIDICA Viral IC Assay; therefore, cross-reactants were diluted patient samples. The test concentration of each substance evaluated at 1 testing site. Three distinct panels containing each two in Negative Control in order to differentiate false positives due to corresponded to three times the researched peak plasma concentration viruses at moderate concentrations (HHV1 & human adenovirus; spectrometry (PCR/ESI-MS) was developed. The assay can reproducibility of the assay at moderate cross-reactivity from background contamination of a human matrix. (Cmax) as recommended per CLSI EP7-A2. Where feasible, some BK polyomavirus and ; Human enterovirus and detect and identify viruses from 13 viral groups, covering positive (3X LOD) test concentrations was Each organism was tested in triplicate. As expected, no false positive substances were pooled to reduce the burden of testing (Table 4b). HHV-5) were run by 3 operators on 3 sets of IRIDICA instruments result was produced in this study. Pooling the substances was done with consideration to avoid combining using 3 lots of Assay Strip on each of 5 days. over 130 viral species. These include herpes simplexviruses 99.6% (Table 6). When clinical plasma specific drugs within a pool which would not be used together in a Number of samples tested Number of samples detected Reproducibility rate Candida albicans HIV HTLV I Human papillomavirus clinical therapeutic setting. This represents a more conservative/worst 1 and 2 (human herpesviruses, HHV-1 and -2), varicella- specimens were analyzed and the results 270 269 99.6% case approach, since multiple drugs are present together. Each Group B Streptococcus Hepatitis A Human rhinovirus * Human enterovirus was not reported as expected for Panel Member 3 on day 4 by one operator. zoster virus (HHV-3), Epstein-Barr virus (HHV-4), compared with standard-of-care RT-PCR substance or pool of substances was tested in triplicate against negative Mycobacterium species Hepatitis virus (HHV-5), (HHV-6 and -7), results, the assay yielded an overall plasma and viral bundles B1, B2, B3, B4, B5, and B6 (Table 4c). The Staphylococcus aureus Hepatitis C virus Mumps virus Kaposi’s sarcoma-associated herpesvirus (HHV-8), calculated positive agreement of 94% viruses were used at approximately three times their limit of detection Staphylococcus epidermidis virus (3X LOD). The seven samples were also tested in triplicate without the polyomaviruses BK and JC, parvovirus B19, adenoviruses (200/212). Following confirmation testing Table 7: Clinical sample performance. The positive percent agreement of the addition of the potentially interfering substances to act as a control to IRIDICA Viral IC Assay was evaluated by testing 212 valid prospectively collected and enteroviruses. Here we characterize the analytical by single-analyte RT-PCR, the overall evaluate any impact on the reported results of the assay. and consented clinical bronchoalveolar lavage (BAL) specimens. 200 specimens Table 4a: Potential interfering substances. were considered to be true positives compared to standard of care results. The sensitivity, analytical specificity, robustness, calculated negative percent agreement For each substance pool and viral bundle combination, the Trade Name Exogenous substance Test conc. negative percent agreement of the IRIDICA Viral IC Assay was evaluated by testing reproducibility, and inclusivity of the method as was 99.6%. identification of viruses reported by the IRIDICA software matched the Aciphex Rabeprazole 60 µg/ml 144 plasma specimens from apparently healthy subjects, yielding 2 false positives reference results for all three replicates. implemented on the IRIDICA™ System,* and compare its Amikin Amikacin 136.8 µmol/l for parvovirus B19, 4 false positives for HHV-4, 1 false positive for HHV-2, and 1 performance to that of traditional single-analyte real time Table 2: Inclusivity study. The inclusivity study verifies the Bactrim Sulfamethoxazole and 1.58 mmol/l Table 4b: Pools of potential interfering substances. false positive for HHV-7 (data not shown). ability of the assay to analytically detect and identify Trimethoprim 138 µmol/l Pool Included Substance(s) PCR detection methods in a collection of deidentified Baraclude Entecavir 24.6 ng/ml multiple strains of the target viruses. A minimum of two 1 Zidovudine (AZT) , Valsartan Number of Number of Test in which Positive Cellcept Mycophenolate Mofetil 102 µg/ml Standard of Care IRIDICA Result Matches Percent (waste) clinical plasma specimens. strains of each virus were tested in this study. Test 2 Abacavir sulfate, Peginterferon alfa-2b, Ribavirin Virus PCR Positive Standard of Care PCR Agreement Cipro Ciprofloxacin 30.2 µmol/l samples were prepared with spikes of the test strains 3 Tenofovir Disoproxil Fumarate, Lamivudine, Ganciclovir, Valganciclovir HCl, Acyclovir Results Result (True Positive) Rate Cleocin Clindamycin 89.1 µmol/l into negative human EDTA plasma. Each test sample 4 Stavudine, Efavirenz, Lopinavir and Ritonavir, Enfuvirtide, Ciprofloxacin Combivir Lamivudine and 4.8 µg/ml virus 1 (HHV-1) 13 13 100 5 Nevirapine, Azithromycin, Valacyclovir HCl was tested in triplicate. Zidovudine 6.0 µg/ml Methods: The QIAamp DSP Virus Kit was used to extract 6 Adefovir, Didanosine, Entecavir, Cidofovir, Mycophenolate Mofetil 2 (HHV-2) 10 9 90 Coumadin Warfarin 32.5 µmol/l Final test conc. 7 Famotidine, Cyclosporine Varicella-zoster virus (HHV-3) 11 11 100 specimens, which were then analyzed using the Viral IC Cyclosporine Cyclosporine 6690 ng/ml Virus ID Strain (copies/ml) 8 Prednisone, Tacrolimus, Azathioprine Assay (08N24-10, CE-IVD).* The limit of detection (LOD) of Cytovene Ganciclovir 31.2 µg/ml Epstein-Barr virus (HHV-4) 15 15 100 Herpes simplex virus 1 1 KOS 2700 9 Atenolol, Amlodipine Besylate, Lisinopril, Rabeprazole a Decadron Dexamethasone 1.53 µmol/l Cytomegalovirus (HHV-5) 45 39 87 (HHV-1) 2 Clinical Isolate (VR-1383) 2700 10 Acetaminophen, Abacavir and Lamivudine, Amikacin, Metronidazole the assay was tested for one isolate from each of the 13 Deltasone Prednisone 0.84 µmol/l 3 HF 2700 11 Dexamethasone, Atazanavir Sulfate, Fluconazole, Ceftazidime Roseolovirus (HHV-6) 20 20 100 4 MacIntyre 2700 Diflucan Fluconazole 245 µmol/l targeted virus groups using Q-PCR quantified virus spiked 12 Dobutamine, Emtricitabine and Tenofovir, Itraconazole, Clindamycin Roseolovirus (HHV-7) 0 0 Herpes simplex virus 2 1 G 600 Diovan Valsartan 192 µg/ml 13 Rapamune, Fosamprenavir, Doxycycline, Piperacillin Kaposi’s sarcoma-associated herpesvirus (HHV-8) 18 17 94 in human plasma. Additionally, 60 diverse isolates were (HHV-2) 2 MS 600 Dobutamine Dobutamine HCl 15000 µg/ml 14 Vinblastine, Lamivudine and Zidovudine, Gentamicin, Vancomycin Varicella-zoster virus 1 Oka 3000 Epivir Lamivudine 6.0 µg/ml BK polyomavirus 26 26 100 tested to confirm the assay’s ability to capture genetic (HHV-3) 2 Clinical Isolate (306640) 1500 15 Warfarin, Amphotericin B, Imipenem, Sulfamethoxazole and Trimethoprim Epzicom Abacavir Sulafte 16.35 µg/ml b 3 82 1500 16 Bilirubin JC polyomavirus 7 4 57 variants. The robustness of the assay was challenged Lamivudine 7.74 µg/ml Epstein-Barr virus (HHV-4) 1 HR-1 600 17 Gamma Globulin Human adenovirus 16 15 94 Flagyl Metronidazole 701 µmol/l through studies of interfering substances, carryover, 2 B95-8 300 18 Hemoglobin Fortaz Ceftazidime 117 µg/ml Human enterovirus 15 15 100 Cytomegalovirus (HHV-5) 1 Merlin 1800 19 Triglycerides potentially cross-reacting organisms, and a reproducibility 2 Davis 28800 Fungizone Amphotericin B 3.3 µg/ml Parvovirus B19 16 16 100 3 AD-169 1800 Fuzeon Enfuvirtide 20.1 µg/ml All viruses 212 200 94.3 analysis using multiple instruments, reagent lots, and Roseolovirus (HHV-6) 1 6A HSB2 GS 1800 Garamycin Gentamicin 21 µmol/l Table 4c: Pools of spiked viruses a 2 6A CBMC SIE 900 Missed CMV detections were confirmed to be samples whose concentration was at or near the IRIDICA Viral Assay LOD. users. The performance of the assay on ill patient Hepsera Adefovir Dipivoxil 73.98 ng/ml Pool spiked virus(es) 3 6A HSB-I 900 b Two of three missed JC detections were in samples that also tested positive for BK virus. These viruses compete for PCR specimens was characterized with 212 de-identified waste 4 6A U1102 900 Imuran Azathioprine 10.8 µmol/l B0 (none) reactants and the lack of JC detection may be due to competition (Table 7). Kaletra Lopinavir and Ritonavir 46.5 µg/ml B1 HHV-3, HHV-4 plasma specimens that had previously tested positive for 5 6A WA 1800 6 6B MT4 HST 900 Lexiva Fosamprenavir 24.9 µg/ml B2 HHV-1, Human adenovirus (serotype 3) Table 8: Summary of discrepant testing. There were 12 false negative samples by the one of the target viruses by standard of care single-analyte 7 6B CBMC MAR 900 Norvasc Amlodipine Besylate 245 nmol/l B3 BK polyomavirus, parvovirus B19, HHV-6 IRIDICA Viral IC Assay. Of the 12 false negative assay results, 6 samples had 8 6B 400 900 Peg-Intron Peginterferon alfa-2b 0.03 µg/ml B4 HHV-2, Human enterovirus (coxsackievirus A9) remaining sample volume sufficient for qPCR testing for discrepancy resolution. real-time at a clinical laboratory. The negative percent 9 6A HSB2 C01 900 Pepcid Famotidine 1.78 µmol/l B5 JC polyomavirus, HHV-5 10 SF 900 Discrepancy resolution of qPCR results are summarized below. Pipracil Piperacillin 1236 µg/ml B6 HHV-8, HHV-7 agreement was assessed by testing 144 plasma specimens Roseolovirus (HHV-7) 1 Supt-1 J1 900 Primaxin Imipenem 249 µg/ml 2 SB 1800 IRIDICA Viral IC Assay IRIDICA Viral IC Re-test qPCR qPCR Result Comments collected from apparently healthy subjects presumed to be Kaposi’s sarcoma-associated 1 Clinical Isolate (B0704) 600 Prinivil Lisinopril 0.74 µmol/l False Negative for Result Quantitation herpesvirus (HHV-8) 2 Clinical Isolate (308222) 600 Prograf Tacrolimus 50 nmol/l negative for the target viruses. Positive detections for any of Table 5a: Carryover study. In order to assess the IRIDICA Viral IC HHV-5 Negative HHV-5 530 copies/ml (1,000 IU/ml) BK polyomavirus 1 Clinical Isolate (VR-837) 600 Retrovir Zidovudine (AZT) 6.0 µg/ml the target viruses were confirmed by standard of care 2 Clinical Isolate (309023) 600 Reyataz Atazanavir Sulfate 20187 ng/ml Assay for carryover, high positive samples were tested adjacent to JC polyomavirus 1 MAD-1 900 Ribavirin Ribavirin 7434 ng/ml negative samples. High positive samples were generated by spiking HHV-5 Negative HHV-5 2438 copies/ml (4,600 IU/ml) single-analyte RT-PCR. 2 MAD-4 900 Sirolimus Rapamune 289.5 ng/ml the viruses listed below into human EDTA plasma. IRIDICA Negative Human adenovirus 1 Type 12 4000 Control was used for the negative sample type. The test viruses HHV-5 BK polyomavirus HHV-5 1219 copies/ml (2,300 IU/ml) 2 Type 18 4000 Sporanox Itraconazole 8388 ng/ml Not tested for BK 3 type 40 4000 Sustiva Efavirenz 49.8 µg/ml chosen, along with the extraction control, exercise all primer pairs of 4 Type 4 4000 Tenormin Atenolol 37.6 µmol/l the IRIDICA Viral IC Assay. HHV-5 HHV-4 HHV-5 954 copies/ml (1,800 IU/ml) Table 1. Confirmed LOD for target viruses. 5 type 1 3200 Truvada Emtricitabin and 7.56 µg/ml Not tested for HHV-4 LOD LOD 6 Type 7A 1600 Tenofovir 1.17 µg/ml JC polyomavirus 468 copies/ml Test Organism 7 Type 14 4000 Virus Spiked Samples Tested concentration position JC polyomavirus BK polyomavirus (copies/ml) (IU/ml) Tylenol Acetaminophen 1324 µmol/l 7 BK polyomavirus 111,000 copies/ml 8 Type 41 4000 BK polyomavirus 6 2 x 10 copies/ml A Herpes simplex virus 1 (HHV-1) 900 N/A Valcyte Valganciclovir HCl 21.39 µg/ml Human adenovirus 700 copies/ml 9 Type 3 12000 7 Valtrex Valacyclovir HCl 24.06 µg/ml HHV-1 12 2 x 10 copies/ml B Human adenovirus BK polyomavirus Herpes simplex virus 2 (HHV-2) 159 N/A Human enterovirus 1 Coxsackievirus A2 384000 BK polyomavirus 7100 copies/ml 2 Echovirus 6 3000 Vancocin Vancomycin 69 µmol/l HHV-5 12 1 x 106 copies/ml A Varicella-zoster virus (HHV-3) 456 N/A 3 Coxsackievirus A1 48000 Vibramycin Doxycycline 67.5 µmol/l 7 HHV-7 6 2 x 10 copies/ml B Epstein-Barr virus (HHV-4) 119 201* 4 Enterovirus 70 48000 Videx Didanosine 150 µg/ml HHV-8 6 1 x 106 copies/ml A Cytomegalovirus (HHV-5) 567 878* 5 Echovirus 11 3000 Vinblastine Sulfate Vinblastine 19.20 µg/ml 7 6 Coxsackievirus B4 3000 Viramune Nevirapine 23.64 µg/ml Human adenovirus 12 2 x 10 copies/ml B Conclusion: The IRIDICA™ Viral IC Assay (Not available in Roseolovirus (HHV-6) 286 N/A 7 Coxsackievirus A4 192000 7 Viread Tenofovir Disoproxil Fumarate 1.17 µg/ml Human enterovirus 12 2 x 10 copies/ml A the USA) is capable of detecting and identifying 13 viral Roseolovirus (HHV-7) 600 N/A 8 Coxsackievirus A17 768000 Vistide Cidofovir 26.1 µg/ml 7 9 Coxsackievirus B3 3000 parvovirus B19 6 2 x 10 copies/ml B groups associated with disseminated viral infections among Kaposi's sarcoma-associated herpesvirus (HHV-8) 200 N/A 10 Coxsackievirus B5 192000 Zerit Stavudine 2046 ng/ml BK Polyomavirus 200 N/A 11 Coxsackievirus A8 96000 Ziagen Abacavir Sulfate 16.35 µg/ml the immuno- compromised. The potential clinical value of 12 Enterovirus 71 3000 Zithromax Azithromycin 15.3 µmol/l JC Polyomavirus 263 N/A SP Carrier configuration 1 SP Carrier configuration 2 13 Enterovirus 69 48000 Zovirax Acyclovir 4.83 µg/ml simultaneously testing for many agents of disseminated viral Human adenovirus (adenovirus 3) 3500 N/A 14 Coxsackievirus A16 192000 2X 2X 1X 1X 2X 2X 15 Coxsackievirus A9 8400 infection rather than using single-analyte tests chosen on the Human enterovirus (Coxsackivirus A9) 2700 N/A Endogenous substances Test conc. Parvorvirus B19 1 Clinical Isolate (306879) 1200 Bilirubin 342 µmol/l A NEG B NEG A NEG NEG B NEG A NEG B parvovirus B19 203 278* 2 Clinical Isolate (306880) 600 basis of empirical judgment warrants further investigation. Gamma Globulin 60 g/l * Individual conversion factors provided by vendor 3 Clinical Isolate (306881) 600 4 Clinical Isolate (306652) 600 Hemoglobin 2 g/l Triglycerides 37 mmol/l Figure 1: Potential carryover events. Positive samples were run adjacent to negative samples, using both configurations shown above. Six independent 24-sample extractions were performed. Each 24-sample isolation produced four carriers of six assay strips each (two carriers for each PCR layout). Each PCR carrier captured five potential carryover events. Therefore, a potential 120 carryover events were tested during this study. * Not available in the USA Copyright © 2015 Ibis Biosciences, an Abbott Company