ZEUS Solinas Borrelia VlsE1/pepC10 IgG/IgM Test System; ZEUS Solinas Borrelia VlsE1/pepC10 IgG/IgM Control Kit

{0} FDA U.S. FOOD &amp; DRUG ADMINISTRATION # 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY AND INSTRUMENT ## I Background Information: A 510(k) Number K230863 B Applicant ZEUS Scientific C Proprietary and Established Names ZEUS Solinas Borrelia VlsE1/pepC10 IgG/IgM Test System; ZEUS Solinas Borrelia VlsE1/pepC10 IgG/IgM Control Kit D Regulatory Information | Product Code(s) | Classification | Regulation Section | Panel | | --- | --- | --- | --- | | LSR, QCH | Class II | 21 CFR 866.3830 - Treponema Pallidum Treponemal Test Reagents | MI - Microbiology | ## II Submission/Device Overview: A Purpose for Submission: To obtain a substantial equivalence determination for the ZEUS Solinas Borrelia VlsE1/pepC10 IgG/IgM Test System and the ZEUS Solinas Borrelia VlsE1/pepC10 IgG/IgM Control Kit. B Measurand: IgM and/or IgG antibodies to Borrelia burgdorferi C Type of Test: Chemiluminescence immunoassay Food and Drug Administration 10903 New Hampshire Avenue Silver Spring, MD 20993-0002 www.fda.gov {1} K230863 - Page 2 of 17 # III Intended Use/Indications for Use: ## A Intended Use(s): See Indications for Use below. ## B Indication(s) for Use: ZEUS Solinas Borrelia VlsE1/pepC10 IgG/IgM Test System The ZEUS Solinas Borrelia VlsE1/pepC10 IgG/IgM Test System uses chemiluminescent immunoassay (CLIA) technology for the qualitative detection of IgG/IgM antibodies to *Borrelia burgdorferi* in human serum. This assay is intended for use on samples from patients with signs and symptoms consistent with or patients suspected of having Lyme disease to assess the presence of IgG/IgM antibodies. Positive results with the ZEUS Solinas Borrelia VlsE1/pepC10 IgG/IgM Test System should be supplemented with additional testing with a Standard two-tier test (STTT) methodology using an IgG and/or IgM *Borrelia burgdorferi* immunoblot assay following current guidelines. Positive supplemental results are supportive evidence of the presence of antibodies and exposure to *Borrelia burgdorferi* and may be used along with patient history, symptoms and other laboratory data to support a clinical diagnosis of Lyme disease. Negative results by the ZEUS Solinas Borrelia VlsE1/pepC10 IgG/IgM Test System should not be used to exclude Lyme disease. The test must be performed on the ZEUS Solinas α instrument. ZEUS Solinas Borrelia VlsE1/pepC10 IgG/IgM Control Kit The ZEUS Solinas Borrelia VlsE1/pepC10 IgG/IgM Control Kit is intended for use as assayed quality control samples to monitor the performance of the ZEUS Solinas Borrelia VlsE1/pepC10 IgG/IgM Test System. The performance characteristics of the ZEUS Solinas Borrelia VlsE1/pepC10 IgG/IgM Control Kit have not been established for any other assays or instrument platforms different from the ZEUS Solinas α instrument. ## C Special Conditions for Use Statement(s): Rx - For Prescription Use Only ## D Special Instrument Requirements: ZEUS Solinas α instrument # IV Device/System Characteristics: ## A Device Description: The ZEUS Solinas VlsE1/pepC10 IgG/IgM Test System uses chemiluminescent immunoassay (CLIA) technology for the qualitative detection of IgG/IgM antibodies to *Borrelia burgdorferi* in human serum. This assay is intended for use on samples from patients with signs and symptoms {2} consistent with or patients suspected of having Lyme disease to assess the presence of IgG/IgM antibodies. The test is performed in the Solinas $\alpha$ instrument. The ZEUS Solinas $\alpha$ instrument is an automated immunoassay analyzer. Analyte detection relies on chemiluminescent technology using acridinium ester-labeled immunoglobulins. The system completely automates mixing of the test reagents and samples in a cuvette and measurement of the resulting luminescent reaction with a luminescent reader. ## B Principle of Operation: The ZEUS Solinas VlsE1/pepC10 IgG/IgM Test on the ZEUS Solinas $\alpha$ instrument uses chemiluminescent reactions for the detection of IgG and IgM antibodies against *Borrelia burgdorferi*. Specifically, serum samples are added to reaction cuvettes, which are loaded into the instrument. First, IgG and IgM antibodies against *Borrelia burgdorferi* in serum samples are bound to antigen-coated magnetic beads (solid phase). After binding, goat-anti-human acridinium-ester labeled conjugate antibodies are added to the reaction to bind to the target antibodies fixed on the solid phase. After several washing steps, trigger solutions are added to the reaction in the signal detection region of the instrument. Then, the reaction cuvettes are moved to the detection chamber where light emitted from the reaction between the trigger solutions and the acridinium ester-labeled conjugate is measured via a photomultiplier and reported by the software as relative light units (RLU). RLU values from each sample are converted to CLIA (Chemiluminescence immune assay) Units via the assay-specific two-point calibration curve. Because the data reduction portion of the Stratec software is unable to use calibrator Unit values containing decimals when calculating the standard curve, the software multiplies the arbitrary Unit values assigned to each calibrator by a factor of 100, which results in the POS/NEG cut-off of 100 Units. The ZEUS Solinas Borrelia VlsE1/pepC10 IgG/IgM Control Kit is intended for use as assayed quality control samples to monitor the performance of the ZEUS Solinas Borrelia VlsE1/pepC10 IgG/IgM Test System. The positive control is optimized to yield an assay signal of approximately 200 to 300 Units (2-3 x the cut off), and the negative control is selected to yield an assay signal of $\leq 50$ Units. The label on each control tube contains a 2D barcode, with an identification number used by the analyzer's software to ensure that each control is matched to the appropriate assay. This matching occurs automatically by the analyzer using the information stored within the control definition module. The controls are designed to assess performance across assay reagent lots, and thus are not assay reagent lot-specific. The performance characteristics of the ZEUS Solinas Borrelia VlsE1/pepC10 IgG/IgM Control Kit have not been established for any other assays or instrument platforms different from the ZEUS Solinas $\alpha$ instrument. ## C Instrument Description Information: 1. Instrument Name: Solinas $\alpha$ 2. Specimen Identification: A camera reader located in the sample loading bay of the instrument can scan barcodes on test tubes for sample identification. K230863 - Page 3 of 17 {3} K230863 - Page 4 of 17 3. Specimen Sampling and Handling: The assay is intended for its use with human serum samples collected by aseptic venipuncture procedures. Fresh and properly stored samples should be used for this test. If testing is not performed within 8 hours from collection, sera may be stored at 2-8 °C for no longer than 3 days. If a delay in testing is anticipated, store test sera at –20°C or lower. Avoid multiple freeze/thaw cycles which may cause loss of antibody activity and give erroneous results. It is the responsibility of the individual laboratory to use all available references and/or its own studies to determine stability criteria for its laboratory. The minimum serum volume required for testing is 100 μL per specimen (10 μL specimen + 90 μL dead volume). For testing in the Solinas α instrument, the sample loading bay is used to load test tubes via a special rack. The sample loading bay has a camera reader to read the information on the 1D or 2D barcode from the test tubes (containing samples such as patient-related specimen, or calibrators, or controls). 4. Calibration: Two assay-specific and lot-specific calibrators are utilized by the Solinas α analyzer. Each calibrator has a barcode containing information such as assay name, lot number, assigned unit value, and expiration date. The Solinas α analyzer automatically tests each calibrator in triplicate and the analyzer's software creates a two-point standard curve using the mean Unit value derived from triplicate analysis of each calibrator. A new calibration is necessary when a new lot of Reagent Cartridge is used, the existing calibration was performed more than eight weeks prior, the Solinas α analyzer underwent servicing, or the values of the ZEUS Solinas Borrelia VlsE1/pepC10 IgG/IgM Controls fall outside of the expected ranges. 5. Quality Control: Quality control testing using the ZEUS Solinas Borrelia VlsE1/pepC10 IgG/IgM Controls should be performed once per day of use or in accordance with local, state and/or federal regulations or accreditation requirements and your laboratory's quality control procedures. It is recommended that the user refers to CLSI document, C24-A3, and 42 CFR 493.1256(c) for guidance on appropriate quality control practices. ZEUS Solinas Borrelia VlsE1/pepC10 IgG/IgM Positive and Negative Controls should be run in singlicate to monitor the assay performance. If control values lie within the expected ranges provided on the certificate of analysis, the test is valid. If control values lie outside the expected ranges, the test is invalid and patient results cannot be reported. Assay calibration should be performed if a control failure is observed and controls and patient specimens must be repeated. The performance of external controls (other than ZEUS Solinas Borrelia VlsE1/pepC10 IgG/IgM Positive and Negative Controls) should be evaluated for compatibility with this assay before they are used. Appropriate value ranges should be established for all external quality control material used by the user. V Substantial Equivalence Information: A Predicate Device Name(s): Zeus ELISA Borrelia VlsE1/pepC10 IgG/IgM Test System {4} B Predicate 510(k) Number(s): K113397 # C Comparison with Predicate(s): | Device & Predicate Device(s): | K230863 | K113397 | | --- | --- | --- | | Device Trade Name | ZEUS Solinas Borrelia VlsE1/pepC10 IgG/IgM Test System | ZEUS ELISA Borrelia VlsE1/pepC10 IgG/IgM Test System | | General Device Characteristic Similarities | | | | Intended Use/Indications For Use | The ZEUS Solinas Borrelia VlsE1/pepC10 IgG/IgM Test System uses chemiluminescent immunoassay (CLIA) technology for the qualitative detection of IgG/IgM antibodies to Borrelia burgdorferi in human serum. This assay is intended for use on samples from patients with signs and symptoms consistent with or patients suspected of having Lyme disease to assess the presence of IgG/IgM antibodies. Positive results with the ZEUS Solinas Borrelia VlsE1/pepC10 IgG/IgM Test System should be supplemented with additional testing with a Standard two-tier test (STTT) methodology using an IgG and/or IgM Borrelia burgdorferi immunoblot assay following current guidelines. | The ZEUS ELISA Borrelia VlsE1/PepC10 IgG/IgM Test System is intended for the qualitative detection of IgG and IgM class antibodies to VlsEl and pepC10 antigens from Borrelia burgdorferi in human serum. The assay is intended for testing serum samples from symptomatic patients or those with a history of Lyme Borreliosis. All positive and equivocal specimens should be tested with a second-tier test such as Western Blot, which if positive, is supportive evidence of infection with B. burgdorferi. Diagnosis of Lyme Borreliosis should be made based on the presence of B. burgdorferi antibodies, history, symptoms, and other laboratory data. Negative first or second tier results should not be used to exclude Borreliosis. This kit is for in vitro diagnostic use. | K230863 - Page 5 of 17 {5} K230863 - Page 6 of 17 | | Positive supplemental results are supportive evidence of the presence of antibodies and exposure to Borrelia burgdorferi and may be used along with patient history, symptoms and other laboratory data to support a clinical diagnosis of Lyme disease. Negative results by the ZEUS Solinas Borrelia VlsE1/pepC10 IgG/IgM Test System should not be used to exclude Lyme disease. The test must be performed on the ZEUS Solinas α instrument. ZEUS Solinas Borrelia VlsE1/pepC10 IgG/IgM Control Kit The ZEUS Solinas Borrelia VlsE1/pepC10 IgG/IgM Control Kit is intended for use as assayed quality control samples to monitor the performance of the ZEUS Solinas Borrelia VlsE1/pepC10 IgG/IgM Test System. The performance characteristics of the ZEUS Solinas Borrelia VlsE1/pepC10 IgG/IgM Control Kit have not been established for any other assays or instrument platforms different from the | | --- | --- | {6} | | ZEUS Solinas α instrument. This kit is for in vitro diagnostic use. | | | --- | --- | --- | | Sample | Human serum | Same | | Measurand | IgG/IgM antibodies | Same | | Antigens | VIsEl and/or pepC10 antigens from Borrelia burgdorferi | Same | | General Device Characteristic Differences | | | | Solid Phase | Polystyrene microscopic magnetic beads | Polystyrene 96 well ELISA plate | | Conjugate | Goat anti-human IgG/IgM labeled with acridinium ester | Goat anti-human IgG/IgM labeled with horse radish peroxidase | | Detection | Chemiluminescence | Colorimetric | | Automation | Fully automated on the Solinasα instrument | Manually performed | | Interpretation criteria | <100 U = negative ≥ 100 U = positive | ≤ 0.9 = negative 0.91 – 1.09 = equivocal ≥ 1.10 positive | VI Standards/Guidance Documents Referenced: Guidance for the Content of Premarket Submission for Software Contained in Medical Devices, Guidance for Industry and Staff, May 2005 CLSI-EP7-A3 VII Performance Characteristics (if/when applicable): A Analytical Performance: 1. Precision/Reproducibility: Precision For the precision study, a total of 48 data points per sample were collected using six serum samples with various levels of antibody concentrations, two calibrators (CAL-1 and CAL-2), and two controls (positive and negative) See Table 1. K230863 - Page 7 of 17 {7} Table 1: Precision Sample Panel | Sample # | Level | Approximate Antibody Concentration | | --- | --- | --- | | 1 | High Positive | ≥ 600 units | | 2 | Medium Positive | 300 units | | 3 | Low Positive | 100 to 150 units | | 4 | Borderline | 85 to 100 units | | 5 | High Negative | 60 to 85 units | | 6 | Low Negative | 0 to 50 units | | ZEUS Solinas Borrelia VlsE1/pepC10 IgG/IgM Negative Control and Positive Control | | | | ZEUS Solinas Borrelia VlsE1/pepC10 IgG/IgM Test System assay Calibrator 1 (CAL-1) and Calibrator 2 (CAL-2). | | | The samples, calibrators, and controls were tested in duplicate, twice per day during twelve separate days using a single Solinas $\alpha$ instrument. Samples, calibrators, and controls were blinded and randomized each day of testing. Results from the precision study are summarized in Table 2 below. Table 2: Precision Study Results | Sample ID | N | Mean | Within Run | | Between Run | | Between Day | | Total | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | | 1 | 48 | 682.8 | 29.1 | 4.3 | 20.5 | 3.0 | 17.0 | 2.5 | 39.5 | 5.8 | | 2 | 48 | 334.5 | 8.4 | 2.5 | 11.9 | 3.6 | 2.5 | 0.7 | 14.8 | 4.4 | | 3 | 48 | 136.2 | 3.5 | 2.5 | 3.2 | 2.3 | 8.5 | 6.3 | 9.7 | 7.1 | | 4 | 48 | 98.9 | 4.7 | 4.8 | 0.0 | 0.0 | 5.1 | 5.1 | 6.9 | 7.0 | | 5 | 48 | 78.1 | 4.3 | 5.6 | 1.9 | 2.5 | 4.8 | 6.2 | 6.8 | 8.7 | | 6 | 48 | 20.4 | 2.8 | 13.9 | 1.9 | 6.4 | 2.8 | 13.7 | 4.4 | 21.7 | | *Neg Control | 48 | 15659 | 1355.0 | 8.7 | 1826.0 | 11.7 | 2016.0 | 12.9 | 3039.0 | 19.4 | | Pos Control | 48 | 228.5 | 8.0 | 3.5 | 7.7 | 3.4 | 0.0 | 0.0 | 11.1 | 4.9 | | CAL-1 | 48 | 124.1 | 4.7 | 3.8 | 1.9 | 1.5 | 4.6 | 3.7 | 6.8 | 5.5 | | CAL-2 | 48 | 69.4 | 3.3 | 4.7 | 1.5 | 2.2 | 3.5 | 5.0 | 5.0 | 7.2 | *Raw RLU data points were utilized since some runs yielded unit values below zero. ## Reproducibility A reproducibility study was conducted over five days across three study sites using the same panel tested for the within laboratory precision study, including lot-specific calibrators and controls. Each sample was tested in triplicate and run twice per day, collecting 30 data points per site for a total of 90 data points per panel member. Each site used a different operator and instrument. Results are summarized in Table 3 below. K230863 - Page 8 of 17 {8} Table 3: Reproducibility Study Results | Sample ID | N | Mean (Units) | Within Run | | Between Run | | Between Day | | Between Site | | Total | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | | 1 | 90 | 658.2 | 83.4 | 12.7 | 0.0 | 0.0 | 10.8 | 1.6 | 90.8 | 13.8 | 84.1 | 12.8 | | 2 | 90 | 351.3 | 40.0 | 11.4 | 0.0 | 0.0 | 0.0 | 0.0 | 40.7 | 11.6 | 40.0 | 11.4 | | 3 | 90 | 113.9 | 7.4 | 6.5 | 2.4 | 2.1 | 1.4 | 1.2 | 4.3 | 3.8 | 7.9 | 6.9 | | 4 | 90 | 91.1 | 8.2 | 9.0 | 3.6 | 3.9 | 0.0 | 0.0 | 1.9 | 2.1 | 8.9 | 9.8 | | 5 | 90 | 76.5 | 10.4 | 13.6 | 0.0 | 0.0 | 3.2 | 4.2 | 9.6 | 12.6 | 10.9 | 14.2 | | 6 | 90 | 22.1 | 9.4 | 42.6 | 0.0 | 0.0 | 0.6 | 2.6 | 10.6 | 48.0 | 9.4 | 42.7 | | Neg Control | 90 | 40.3 | 6.5 | 16.2 | 0.0 | 0.0 | 0.8 | 2.0 | 7.2 | 17.9 | 6.6 | 16.3 | | Pos Control | 90 | 298.9 | 41.3 | 13.8 | 0.0 | 0.0 | 3.6 | 1.2 | 44.8 | 15.0 | 41.4 | 13.9 | | CAL-1 | 90 | 123.8 | 11.9 | 9.6 | 0.0 | 0.0 | 2.1 | 1.7 | 12.3 | 10.0 | 12.1 | 9.8 | | CAL-2 | 90 | 84.4 | 5.6 | 6.6 | 0.9 | 1.1 | 1.5 | 1.8 | 4.6 | 5.4 | 5.8 | 6.9 | *Raw RLU data points were utilized since some runs yielded unit values below zero. 2. Linearity: N/A 3. Interference/Cross-Reactivity: Interference Endogenous substances identified below (Table 4) were assessed for potential interference by testing a serum sample (90-100 units) in the presence of each of the substances. The samples spiked with interferents were tested in triplicate. The percent recovery for each interferent was calculated using the equation: Percent Recovery = (Mean unit value from three replicates of each sample spiked with an interferent/Mean unit value from the three replicates of each corresponding control sample) x100. Table 4: Interferents Testing Results | Interferent | Testing concentration | Solvent/Control | Percentage Recovery | | --- | --- | --- | --- | | Hemoglobin | 10 mg/mL | N/A | 83% | | Triglycerides | 15 mg/mL | Methanol | 106% | | Bilirubin | 0.4 mg/mL | PBS | 106% | | Albumin | 120 mg/mL | PBS | 93% | | Cholesterol | 5 mg/mL | Methanol | 111% | | Rheumatoid Factor (Rf) | 100 IU/mL | PBS | 98% | An interference effect was initially observed for hemoglobin at $10\mathrm{mg / mL}$ which resulted in a lower percent recovery. Additional testing was performed to confirm the effect and identify the hemoglobin concentration where this effect would disappear. However, lower than expected recovery was not observed at $10\mathrm{mg / mL}$ hemoglobin during repeat testing. Results are shown in Table 5 below. K230863 - Page 9 of 17 {9} Table 5: Repeat Evaluation of Hemoglobin Interference | Sample Concentration | CLIA Units | Percentage Recovery | | --- | --- | --- | | | Mean of 3 replicates | | | Positive Control | 102.28 | N/A | | 10mg/mL | 96.28 | 94.13% | | 7.5 mg/mL | 89.83 | 87.13% | | 5 mg/mL | 94.25 | 92.15% | | 2.5 mg/mL | 98.63 | 96.43% | | 1mg/mL | 97.64 | 95.46% | Cross-Reactivity A total of 246 samples containing antibodies against 23 potentially cross-reactive infections and conditions were tested. Results are summarized in Table 6 below. K230863 - Page 10 of 17 {10} Table 6: Cross-reactivity Study Results | Category | Tested Samples (n) | Positive | | --- | --- | --- | | Tick-borne | | | | Babesiosisa | 20 | 4 | | Autoimmune/Rheumatoid | | | | Anti-Nuclear Antibodies (ANA) Serological | 10 | 0 | | Systemic Lupus Erythematosus (SLE) | 10 | 0 | | Sjogrens Syndrome | 10 | 0 | | Rheumatoid Arthritisb | 30 | 1 | | Rheumatoid Factore | 15 | 0 | | Viral | | | | Cytomegalovirus (CMV)d | 10 | 0 | | Epstein-Barr Virus (EBV) EA | 10 | 0 | | Epstein-Barr Virus (EBV) EBNA IgG | 10 | 0 | | Epstein-Barr Virus (EBV) VCA IgMb | 9 | 2 | | Epstein-Barr Virus (EBV) VCA IgG | 10 | 0 | | Human Immunodeficiency Virus (HIV) | 5 | 0 | | Influenza Virus (Flu A) | 10 | 0 | | Influenza Virus (Flu B) | 10 | 0 | | Parvovirus IgM | 7 | 0 | | Parvovirus IgG | 10 | 0 | | Bacterial | | | | H. influenza b | 10 | 0 | | H. pylori | 10 | 0 | | B. pertussis | 10 | 0 | | Legionella | 10 | 0 | | T. pallidumb | 20 | 3 | | Miscellaneous | | | | Human anti-mouse antibodies | 5 | 0 | | Cancer | 10 | 0 | aAll 4 samples positive by the VlsE1/pepC10 Solinas CLIA test were also positive by the predicate ELISA and Borrelia IgG and IgM immunoblots bSamples positive on the VlsE1/pepC10 CLIA assay were negative by Borrelia IgG and IgM immunoblots c15 of the 30 Rheumatoid Arthritis samples were positive for Rheumatoid Factor (RF) IgM. 0 of the 15 RF positives were positive on the VlsE1/pepC10 Solinas CLIA test dThe 10 CMV samples consisted of 8 IgG and 2 IgM positive samples 4. Assay Reportable Range: N/A 5. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods): **Specimen stability** Stability of specimens stored at both room temperature and in refrigeration (2-8°C) were tested by using high positive, low positive and negative serum samples. One group of samples was aliquoted and tested in triplicate after storage at room temperature and a second group was tested in triplicate after storage in refrigeration. Subsequent measurements for both conditions were conducted at 48, 96, and 168 hours. Samples stored in refrigeration (2-8°C) demonstrate stability for up to 3 days. Results are summarized in Table 7 below. K230863 - Page 11 of 17 {11} Table 7: Sample Stability Study Results | Storage Time | Storage Condition | Result | Sample ID | | | | --- | --- | --- | --- | --- | --- | | | | | Mean High Positive | Mean Low Positive | Mean Negative | | T=0 | Baseline | Units | 1049 | 136 | 39 | | 1 Day | Room Temperature | Units | 985 | 131 | 37 | | | | % of T = 0 | 94 | 96 | 95 | | | 2-8°C | Units | 1049 | 140 | 38 | | | | % of T = 0 | 100 | 103 | 98 | | 3 Days | Room Temperature | Units | 951 | 136 | 39 | | | | % of T = 0 | 91 | 100 | 100 | | | 2-8°C | Units | 1064 | 148 | 38 | | | | % of T = 0 | 101 | 109 | 98 | | 7 Days | Room Temperature | Units | 1036 | 146 | 39 | | | | % of T = 0 | 99 | 107 | 102 | | | 2-8°C | Units | 1098 | 151 | 40 | | | | % of F/T 1 | 105 | 111 | 104 | | 14 Days | Room Temperature | Units | 1035 | 151 | 41 | | | | % of T = 0 | 99 | 111 | 105 | | | 2-8°C | Units | 1064 | 159 | 41 | | | | % of T = 0 | 101 | 117 | 106 | # Calibrators Two lot-specific calibrators are provided with each assay kit. The calibrators are used to construct a two-point calibration curve with two signals: the first calibrator optimized to yield a signal of approximately 120 CLIA Units and the second calibrator optimized to yield an assay signal of approximately 80 CLIA Units. These unit values are approximately $20\%$ above and $20\%$ below the assay cut-off of 100 Units. During the calibration process, the instrument assays each calibrator in triplicate, and the software creates a two-point calibration curve using the mean RLU value and mean Unit value of each calibrator. The calibration curve is stored within the software and is used to calculate results for patients and controls. Calibrator frequency was determined by running eight samples and two controls in singlicate, once per week, over the course of nine weeks after the calibration event. Acceptance was determined by a variability not greater than $10\%$ for all samples, except High Pos, Medium/High Neg, Low Neg and Low Neg (NC) where variability is expected to be higher. A single calibration event every eight weeks was determined to be acceptable (Table 8). K230863 - Page 12 of 17 {12} Table 8: Calibration Frequency Study Results | Sample | Category | Day1 | Week 1 | Week 2 | Week 3 | Week 4 | Week 5 | Week 6 | Week 7 | Week 8 | Week 9 | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | Units | % Day 1 Units | | | | | | | | | | 1 | High Pos | 1002 | 113 | 112 | 113 | 114 | 110 | 109 | 108 | 103 | 101 | | 2 | Medium/High Pos | 659 | 98 | 98 | 103 | 106 | 96 | 96 | 100 | 93 | 89 | | 3 | Medium/High Pos | 605 | 100 | 96 | 97 | 100 | 96 | 94 | 96 | 96 | 95 | | 4 | Low/Med Pos | 333 | 87 | 95 | 99 | 92 | 95 | 97 | 94 | 93 | 94 | | 5 | Low Pos | 124 | 109 | 96 | 106 | 106 | 101 | 100 | 93 | 98 | 100 | | 6 | Borderline/High Neg | 86 | 108 | 103 | 104 | 105 | 97 | 95 | 94 | 107 | 98 | | 7 | Med/ High Neg | 69 | 118 | 100 | 104 | 101 | 112 | 99 | 90 | 103 | 88 | | 8 | Low Neg | 21 | 103 | 59 | 52 | 77 | 88 | 67 | 61 | 136 | 74 | | NC | Low Neg | 4 | 82 | 52 | 130 | 195 | 91 | 80 | 38 | 116 | 112 | | PC | Low/Med Pos | 203 | 101 | 97 | 106 | 103 | 99 | 105 | 103 | 106 | 93 | | CAL1 | Low Pos | 114 | 114 | 100 | 109 | 97 | 102 | 102 | 97 | 102 | 96 | | CAL2 | High Neg | 65 | 110 | 103 | 109 | 108 | 115 | 96 | 98 | 101 | 96 | # Controls An assay-specific control kit is provided separately from the assay reagent kit. The control kit contains a positive control vial (1 mL) and a negative control vial (1 mL). During the manufacturing process, the positive control is optimized to yield an assay signal of approximately 200 to 300 Units (2-3X the cut off), and the negative control is selected to yield an assay signal of $\leq 50$ Units. Each control tube label contains information such as the control expiration date, lot number, and recommended storage conditions. The label also has a 2D barcode, which contains an identification number used by the analyzer's software to match the control to the appropriate assay. This matching occurs automatically by the analyzer using the information stored within the control definition module. The controls are designed to assess performance across assay reagent lots, and thus are not assay reagent lot-specific. The information from the certificate of analysis for each new lot of controls must be entered into the control definition module within the analyzer's software. The information entered into the control definition module include the assay name that each control is assigned to, the control lot number, the control expiration date, the number of replicates to be assayed during each control run, the acceptable control lot-specific Unit value ranges, and the maximum allowable time period between control runs. For the ZEUS Solinas VlsE1/pepC10 IgG/IgM Test System, a control run will be required every 24 hours. # Control process During the sample loading process and the 2D barcode of each control is scanned, and a sample rack position is assigned for each control within the analyzer's software. The analyzer automatically assays each control in singlicate, and the software calculates a Unit value for each control. Then, the software determines if each control is valid, based upon the lot-specific ranges present within the corresponding control definition. If the controls yield valid results, then patient results can be reported until the next control run is requested by the analyzer's software (24 hours). However, if the controls yield invalid results, then the patient results cannot be reported until a valid control run has been achieved. # 6. Detection Limit: N/A K230863 - Page 13 of 17 {13} 7. Assay Cut-Off: A preliminary assay cut-off was established by testing 200 apparently healthy donor samples from endemic and non-endemic regions and calculating the mean and Standard Deviation of the RLU values. The preliminary assay cut-off was further adjusted after evaluation of the CDC 92-sample Lyme Research Panel II. 8. Accuracy (Instrument): Not applicable. 9. Carry-Over: Carry over contamination in the Solinas $\alpha$ instrument was evaluated using 36 high positive ($\geq$ 600 units) and 36 negative samples ($\leq$50 units). Additionally, lot specific calibrators and the ZEUS Solinas Borrelia VlsE1/pepC10 IgG/IgM controls were included in the study. Briefly, positive and negative samples were alternated and sequentially assayed using the ZEUS Solinas Borrelia VlsE1/pepC10 IgG/IgM Test System. Acceptance criteria was determined by a within run CV of 15% or less for positive samples, and a CV of 50% or less for the negative samples. A summary of the study results is presented in Table 9 below. Table 9: Carry-Over Study Results | Test Level | Number of Positive Panels Percentage | %CV Units | | --- | --- | --- | | High | 36/36 | 3.8 | | Positive | 100% | | | Negative | 36/36 | 16.7 | | | 100% | | B Comparison Studies: 1. Method Comparison with Predicate Device: Method comparison using CDC Serum Panel The 280 samples of the CDC Pre-Marketing Borrelia panel were tested using the ZEUS Solinas Borrelia VlsE1/pepC10 IgG/IgM Test System and the ZEUS ELISA Borrelia VlsE1/pepC10 IgG/IgM Test System. This panel consisted of 60 cases of early Lyme disease (LD), 3 cases of cardiac LD, 7 cases of neurologic LD, 20 cases of late LD, 100 healthy controls and 90 non LD, diseased controls. The samples were tested, and their results were compared. Results for first and second tier testing comparisons can be seen in Tables 10 and 11 below, respectively. K230863 - Page 14 of 17 {14} Table 10: CDC Pre-market panel – First Tier Testing Results | Sample Category (N) | ZEUS VlsE1/pepC10 CLIA (Test) | | | | ZEUS VlsE1/pepC10 ELISA (Predicate) | | | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | Pos | Neg | Sens/Spec | 95% CI | Pos or Equiv | Neg | Sens/Spec | 95% CI | | Early Lyme (N=60) | 48 | 12 | 80.0% | (68.22 – 88.17) | 50 | 10 | 83.33% | (71.97 – 90.69) | | Cardiac Lyme (N=3) | 2 | 1 | 66.67% | (20.77 – 92.85) | 3 | 0 | 100% | (43.85 – 100) | | Neurologic Lyme (N=7) | 7 | 0 | 100% | (64.57 – 100) | 7 | 0 | 100% | (64.57 – 100) | | Late Lyme (N=20) | 20 | 0 | 100% | (83.89 – 100) | 20 | 0 | 100% | (83.89 – 100) | | Healthy Controls (N=100) | 2 | 98 | 98.0% | (93 – 99.45) | 4 | 96 | 96.0% | (90.16 – 98.43) | | Disease Controls (N=90) | 11 | 79 | 87.78% | (79.43 – 93.04) | 7 | 83 | 92.22% | (84.88 – 96.18) | Additionally, samples were tested using the Gold Standard IgM and IgG immunoblots as the second-tier test for both first-tier methods, following a standard two-tier method. Results are shown in Table 11, below. Table 11: CDC Pre-market panel – Standard Two-Tier Testing Results | Sample Category (N) | Test Assay + Line Blots IgG or IgM | | | | Predicate Assay + Line Blots IgG or IgM | | | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | Pos | Neg | Sens/Spec | 95% CI | Pos or Equiv | Neg | Sens/Spec | 95% CI | | Early Lyme (N=60) | 43 | 17 | 71.67% | (59.23 – 81.49) | 41 | 19 | 68.33% | (57.77 – 78.69) | | Cardiac Lyme (N=3) | 2 | 1 | 66.67% | (20.77 – 92.85) | 2 | 1 | 66.67% | (20.77 – 92.85) | | Neurologic Lyme (N=7) | 7 | 0 | 100% | (64.57 – 100) | 7 | 0 | 100% | (64.57 – 100) | | Late Lyme (N=20) | 20 | 0 | 100% | (83.89 – 100) | 20 | 0 | 100% | (83.89 – 100) | | Healthy Controls (N=100) | 0 | 100 | 100% | (96.3 – 100) | 0 | 100 | 100% | (96.3 – 100) | | Disease Controls (N=90) | 0 | 90 | 100% | (95.91 – 100) | 0 | 90 | 100% | (95.91 – 100) | ## Multi-site Prospective Study A study was executed to compare the performance of the ZEUS Solinas Borrelia VlsE1/pepC10 IgG/IgM Test System to the predicate ZEUS ELISA Borrelia VlsE1/pepC10 IgG/IgM Test System using prospectively collected patient samples. This study was conducted at three different Lyme disease endemic locations in the U.S. with three different Solinas $\alpha$ instruments and technicians. Sites collected routine, left-over, de-identified serum samples submitted to their respective facilities for routine Lyme disease serology testing. Each site collected 600 samples, adding to a total of 1800 samples. Samples collected in each site were divided into three groups of 200 and redistributed among three testing sites, each testing site receiving a total of 600 samples representing all three collection sites. All samples were first tested using a first-tier method using both the ZEUS Solinas CLIA assay and the predicate. Any sample that generated a positive result by ZEUS Solinas VlsE1/pepC10 IgG/IgM Test System or a positive/equivocal result by ZEUS ELISA VlsE1/pepC10 IgG/IgM Test System was then tested in a Standard two-tier Testing method using the $B$. burgdorferi IgG and IgM Gold Standard immunoblots at ZEUS Scientific. One specimen was excluded from any data analyses as it was determined to be invalid according to the Gold Standard Diagnostics labeling due to excessively high background staining on the IgM immunoblot. Negative samples in the first-tier testing were not further tested. Results from K230863 - Page 15 of 17 {15} the first-tier testing are described in Table 12 below and results of the Standard Two-Tiered Testing method are described in Table 13 below. Table 12: Prospective Study - First Tier Test Results Compared to Predicate | | ZEUS ELISA Borrelia VlsE1/pepC10 IgG/IgM Test System (Predicate Assay) | | | | | --- | --- | --- | --- | --- | | | | Positive | Equivocal | Negative | | ZEUS Solinas Borrelia VlsE1/pepC10 IgG/IgM Test System | Positive | 186 | 10 | 112 | | | Negative | 29 | 10 | 1452 | | | Total | 215 | 20 | 1799 | PPA: 86.51% (196/235) 95% CI: 81.3 – 90.44% NPA: 92.84% (1452/1564) 95% CI: 90.23 – 92.80% Table 13: Prospective Study – Standard Two-Tier Test Results Compared to Predicate | | ZEUS ELISA Borrelia VlsE1/pepC10 IgG/IgM Test System (Predicate Assay) with Immunoblot (STTT) | | | | --- | --- | --- | --- | | | | Positive | Negative | | ZEUS Solinas Borrelia VlsE1/pepC10 IgG/IgM Test System with Immunoblot (STTT) | Positive | 160 | 25 | | | Negative | 7 | 1614 | | | Total | 167 | 1799 | PPA: 95.81% (160/167) 95% CI: 91.6 – 97.95% NPA: 98.47% (1607/1632) 95% CI: 97.75 – 98.96% Comparing the two methods in a STTT method using immunoblot resulted in PPA and NPA point estimates of 95.8% and 98.5%, respectively. Cumulatively these results demonstrate substantial equivalence to the predicate device. 2. Matrix Comparison: N/A C Clinical Studies: 1. Clinical Sensitivity and Specificity: N/A 2. Other Clinical Supportive Data (When 1. and 2. Are Not Applicable): N/A D Clinical Cut-Off: N/A E Expected Values/Reference Range: The total number of samples tested and the number of positive samples for each population in the studies conducted to establish the performance characteristics of the ZEUS Solinas Borrelia VlsE1/pepC10 IgG/IgM are summarized in Table 14 below. K230863 - Page 16 of 17 {16} Table 14: Summary of Prospective Specimen Cohort | Populations | | Number tested | Positive/Tested | | --- | --- | --- | --- | | Prospective Studies | | 1799 | 186/1799 | | Characterized Sample Study | Lyme disease | 90 | 77/90 | | | Negative | 190 | 13/190 | | Endemic Controls | | 100 | 1/100 | | Non-Endemic Controls | | 100 | 1/100 | F Other Supportive Instrument Performance Characteristics Data: N/A VIII Proposed Labeling: The labeling supports the finding of substantial equivalence for this device. IX Conclusion: The submitted information in this premarket notification is complete and supports a substantial equivalence decision K230863 - Page 17 of 17