Affected DAD discs include manufacturing kit lot numbers through current inventory with a distribution date of September 16, 2015.

Transcription

1 IMPORTANT: CUSTOMER CORRECTION NOTICE Direct Amplification Discs included with kits: Simplexa HSV 1 & 2 Direct Simplexa Flu A/B & RSV Direct Simplexa Group A Strep Direct February 10, 2016 Dear Customer/Distributor, The purpose of this letter is to advise you that Focus Diagnostics is providing a customer correction notice for the Direct Amplification Discs (DAD) supplied with the Simplexa HSV 1 & 2 Direct (MOL2150), Simplexa Flu A/B & RSV Direct (MOL2650) and Simplexa Group A Strep Direct kits (MOL2850). ISSUE: Focus Diagnostics has received customer complaints regarding MOL2150 and MOL2650 kits. Two issues have been reported: 1) First with early Cycle threshold (Ct) in the sample wells of the Direct Amplification Discs (MOL1455, MOL1452, MOL1451) while performing the MOL2150 and MOL2650 testing (see Figure 1 for some examples of early Ct). This error may result in a false negative result, a false positive result or Error Code(s) 500 or ) Additionally, some customers are reporting insufficient specimen volume errors which may result in higher invalid rates. In those cases, the error results in a Report Note Statement of Insufficient Specimen Volume. At this time, we do not know if the two issues are related. Affected DAD discs include manufacturing kit lot numbers through current inventory with a distribution date of September 16, To date, we received a total of 24 complaints from 12 customers for the early Ct issue and 20 complaints from 9 customers for the Insufficient Specimen Volume error code. Three customers reported both issues. RECOMMENDATION: In our internal testing, we were able to reproduce the early Ct threshold issue only after DAD discs were reused after a partial run. Therefore, Focus Diagnostics is not requesting removal of the kits or Direct Amplification Discs from the test facility; however we are requesting that you only run a full DAD disc or discard the DAD disc after a partial run. Focus will provide extra DAD discs without charge until the issues are resolved. Until further notice, please do not reuse the DAD disc from these kits.

2 If an insufficient specimen volume error is obtained with any of the identified kits, Focus Diagnostics requests the customer to repeat any invalid results using a new disc and follow the instructions stated in the package inserts for re-testing INVALID RESULTS. If upon re-test, the problem is unresolved or you experience an increase in invalid results, please contact our Technical Services department. As noted in the Focus Package Inserts (Section: Limitations), results from these tests must be considered in conjunction with the clinical history, epidemiological data, and other data available to the clinician evaluating the patient. We are currently investigating these issues with our supplier (3M Company).We will provide further recommendations as we determine further corrective steps to rectify the issues. MOL1455 is supplied with each MOL2150, MOL2650 and MOL2850 kit. Three discs of MOL1452 are packed in a box and labeled as MOL1455. Additionally, a box of 24 discs of MOL1452 is labeled as MOL1451. Affected DAD disc numbers include manufacturing kits through current inventory with a manufacturing date of July 30, 2015, and a distribution date of September 16, Please evaluate your inventory to verify if you have impacted DAD lots and do not reuse DAD discs. For any Insufficient Volume Errors; retest with a new DAD disc. RISK Potential for false positive result* *In any of these risk sections, for all cases where other medications are prescribed, please refer to the package inserts of those medicines for their effects. Simplexa HSV 1 & 2 Direct (MOL2150) A false positive in a patient with presumed HSV-encephalitis will continue to receive IV acyclovir, which may result in a prolonged therapy and hospitalization. Additionally, the true cause of meningitis/encephalitis may be missed or delayed, which, if requiring a different therapeutic course, could pose a risk of harm to the patient (e.g. if other medications are discontinued). A false positive in a patient with genital infection could lead to psychosocial stress and may increase the risk of acquiring HSV, if patient no longer follows safe practices with HSV discordant partners. Pregnant patients may not receive strong HSV prevention counseling, if antibody test for HSV2 are positive (highest risk to baby is primary infection in last trimester). Patients may receive antivirals if PCR positive; with potential drug side effects, and/or costs. Simplexa Flu A/B & RSV Direct (MOL2650) A false positive is little risk in most patients, as supportive treatment is low risk and should be given appropriately based on symptoms, not lab findings; however it may lead to unnecessary administration of antiviral medications. A false positive for possible RSV cases in the immunocompromised population may lead to antivirals and passive immunotherapy for pediatric patients, but this would be rare in adults except for certain categories, like immunocompromised hosts (i.e., bone marrow transplant patients) who may be at high risk for pneumonia and death from RSV. It may lead to cohorting of the patient with true RSV positive patients, leading to increased risk of infection in the patient with the false positive result. The health care provider may stop looking for other causes of respiratory illness, and if wrong, may miss the real underlying cause of the individual symptoms and not treat appropriately. Diagnosis should be made in light of the entire clinical picture, including retesting, if at high risk.

3 Simplexa Group A Strep Direct (MOL2850) A false positive may result in unnecessary antibiotic use with numerous potential side effects. Potential risks of antibiotic use, include; gastrointestinal reactions, allergic reactions (rash and more serious anaphylaxis) and C. difficile colitis. If the result does not align with clinical symptoms; it is suggested to repeat testing and confirm with culture. RISK Potential for false negative result* Simplexa HSV 1 & 2 Direct (MOL2150) A false negative in a patient with presumed HSV encephalitis may lead to premature discontinuation of acyclovir and the patient might worsen. This is especially important if treatment is for HSV encephalitis. If acyclovir is discontinued for HSV meningitis, symptoms might worsen but will not result in permanent sequelae or significant risk to patient other than temporary discomfort (headache, photophobia etc.) A false negative in a sexually active patient with genital infection may lead to the patient not receiving as strong counseling, or lead to false sense of security, and continued high risk behavior leading to adverse outcome (e.g., an increased susceptibility to HIV). There may be increased risk of transmitting infection to sexual partners if the patient doesn t follow safer sex practices. If in pregnant patient herself, may decrease vigilance for evaluation for HSV lesions at time delivery which could put the infant at risk if active infection is missed at delivery. Primary infection acquired during pregnancy, if clinically recognized, may lead to additional interventions, including consideration for retesting. Simplexa Flu A/B & RSV Direct (MOL2650) A false negative presents little risk as illness is typically self-limited and treatment is essentially supportive. If a false negative were to lead to delay of medication, impact is typically not substantial as antiviral therapy simply reduces the duration of illness in most cases. Antiviral therapy can reduce complications and mortality in severe cases and a false negative may hinder timely administration of these medications, but in most cases, antivirals would be given empirically in severe cases regardless of test results. Simplexa Group A Strep Direct (MOL2850) A false negative would slightly prolong symptoms without antibiotics; however there is a theoretical risk of post Streptococcus rheumatic fever and a low potential risk of rare strep throat complications such as peritonsillar abscess, bacteremia etc. If the result does not align with clinical symptoms; it is suggested to repeat testing and confirm with culture. RISK Potential for delayed result* Simplexa HSV 1 & 2 Direct (MOL2150) In a patient infected with HSV encephalitis, a delayed result would have minimal impact if the patient was responding to acyclovir. For HSV non-infected patients, a delayed result may be continuation of acyclovir. For HSV meningitis, a delayed result would possibly mean that acyclovir is not started and that symptoms might be prolonged. HSV meningitis can look like other benign, and untreatable viral infections (e.g. enteroviral meningitis) so empiric acyclovir treatment is not usually given, unless patient has obvious HSV2 primary genital outbreak, which might increase suspicion of HSV.

4 In a patient with genital infection, a delayed result may lead to patient anxiety, the potential transmission to partner if infected, and could impact potential evaluation of pregnant woman in a timely manner. Simplexa Flu A/B & RSV Direct (MOL2650) A delayed result would pose little risk as illness is typically self-limited and treatment is essentially supportive. If a result were delayed, this may lead to delay of antiviral medication, but the impact is not substantial. Antiviral therapy can reduce complications and mortality in severe cases, a delayed result may hinder timely administration of these medications; however, in a critically ill patient, antivirals will be administered empirically, regardless of testing delays. Simplexa Group A Strep Direct (MOL2850) An invalid or delay of 1-9 days is unlikely to have significant patient impact as treatment only decreases symptoms for about 24 hours. If delay is more than 9 days, it may theoretically increase risk of rheumatic fever, which to prevent, should receive antibiotics within 9 days of pharyngitis. As with all cases of disease diagnostics, the results from these kits must be considered in conjunction with the clinical history, epidemiological data and other laboratory information available to the clinician evaluating the patient. ACTIONS BY THE CUSTOMER/DISTRIBUTOR: Check to see if you have any of the identified MOL1455, MOL1452 and MOL1451 products. Only run a full DAD disc or discard the DAD disc after a partial run Based on the physician s assessment of the patient, if questions still arise, additional testing may be necessary. For Insufficient Volume Errors, if upon re-test and the problem is unresolved, please contact Focus Technical Services department. Acknowledge that you have received this notification by signing the enclosed acknowledgement form and the form to Technicalinfo@focusdx.com or fax back to Focus Diagnostics Technical Services at within 10 business days. Distributors only: As part of our Quality System we may audit your facility to ensure activities assigned to your facility are properly conducted. If selected, we will contact you prior to scheduling the audit. See attached Direct Amplification Disk labels and product information for ease in identifying the product. Please refer to the lot number adjacent to the lot symbol LOT. Please accept our apologies for any inconvenience this may have caused. If you have any questions or require additional information or DAD discs, please contact our Technical Services department at , select option 3, between the hours of 7am to 5pm (PST) or send an to Technicalinfo@focusdx.com Adverse reactions or quality problems experienced with the use of this product may be reported to FDA or appropriate International Ministry of Health: or Call FDA FDA-1088

5 Sincerely, Valerie Cimmarusti Vice President, Quality, Regulatory and Clinical Affairs Attachments: Acknowledgement Form Figure 1: Early Ct examples Direct Amplification Disc Labels Simplexa HSV 1 & 2 Direct Package Insert Simplexa Flu A/B & RSV Direct Package Insert Simplexa Group A Strep Direct Package Insert

6 CORRECTION RETURN RESPONSE Acknowledgement and Receipt Form - Response required Direct Amplification Discs included with kits: Simplexa HSV 1 & 2 Direct Simplexa Flu A/B & RSV Direct Simplexa Group A Strep Direct I have read and understand the correction instructions dated February 10, 2016 Yes No Were there any adverse events associated with the product? Yes No If yes, please explain: ACKNOWLEDGEMENT: Signature of Recipient: Date: Name: Title Telephone address Please the form to Technicalinfo@focusdx.com or fax back to Focus Diagnostics Technical Services at within 10 business days.

7 DAD Correction Notice dated February 10, 2016 Figure 1 Early Ct examples:

8

9 Label Attachments: Label for Direct Amplification Disc Kit MOL1455 (Package of 3 DAD): Label for Direct Amplification Disc MOL1452 (Individual DAD):

10 Label for Direct Amplification Disc MOL1451 (Package of 24 DAD):

11 IMPORTANT: CUSTOMER CORRECTION NOTICE - Amendment Direct Amplification Discs included with kits: Simplexa HSV 1 & 2 Direct Simplexa Flu A/B & RSV Direct Simplexa Group A Strep Direct February 11, 2016 Dear Customer/Distributor, Focus is providing an amendment to the Customer Correction Notice dated February 10, To clarify what is considered an early Ct, Focus would consider Internal Control (IC) Ct s less than 25 as early Ct. If an internal control Ct is less than 25, additional testing may be necessary. The following are images that demonstrate where Focus lot numbers appear and 3M lot numbers appear. This is followed by a table that cross-references the lot numbers for further clarification. Direct Amplification Disc Kit (3 pack) Focus Lot # MOL1455

12 Direct Amplification Disc (24 pack) Focus Lot # MOL1451 3M Lot # 3M Lot #

13 3M Manufacturing Lot # (Pouch and White Box of 24 DAD) Focus Lot # MOL 1455 Box of 3 DAD (3 MOL1452) Focus Lot # (green sticker on shipping box) MOL 1451 Box of 24 DAD (24 MOL1452) n/a / / / / Sincerely, Valerie Cimmarusti Vice President, Quality, Regulatory and Clinical Affairs

14 IMPORTANT: CUSTOMER CORRECTION NOTICE Amendment(2) Direct Amplification Discs included with kits: Simplexa HSV 1 & 2 Direct Simplexa Flu A/B & RSV Direct Simplexa Group A Strep Direct February 15, 2016 Dear Customer/Distributor, Focus is providing an amendment to the Customer Correction Notice dated February 10, To clarify what is considered an early Ct, Focus would consider Internal Control (IC) Ct s less than 25 as early Ct. If an internal control Ct is less than 25, additional testing may be necessary based on the treating physician's clinical assessment of their patient. To determine if you had instances of early Ct s less than 25, please refer to Section III Analyzing Runs, pages 6-10 and 6-16 in the Focus Diagnostics, Operator Manual for Integrated Cycler Studio 6.0 For Use with IVD Assays First, we suggest you screen the Internal Control (IC) channel. The IC Ct value of 25 or less would indicate you should take a closer look at the result in that wedge. If you want to take a closer look at each wedge, review the steps below. Steps to analyze Ct values: 1. Click the print button to view the report 2. Check the Include Ct values box 3. Review Q670 channel (Internal Control) or inspect all channels to see each wedge Cts 4. Click close For a more detailed analysis, please view excerpts from the ICS Operator Manual covering Section III, pages 6-10 and 6-16 below:

15 Section III: Page 6-10 Analyzing Runs 1 When the disc processing is complete, the message Analysis Complete appears in the Run Status field and the results are displayed on the screen. 2 Click the Print button to print a full report of the results. 3 To view a detailed analysis of the completed run, click the Details button for any well in the disc matrix. Analyzing Runs

16 4 The Data tab screen displays the date and time of the run, the disc ID, total run time, and the lot ID. Enable or disable the checkboxes in the Dyes field to view a plot of dyes on the graph. 5 The Details tab screen displays the instrument ID, the disc ID, the spectral matrix, and any pertinent notes for this run. A results summary includes fluid checks, Ct values, and any sample failures which are highlighted in yellow. 6 Click the Print button to print a full report of the results. Section III Generating a Segment Report Page 6-15 Printing Run segments can be printed or saved as files.

17 Generating a Segment Report 1 From the analysis screen, select the segment to print. 2 Select/de-select any wedges to include as graphs by checking/unchecking the box next to the wedge. Note: Data for all disc wedges prints, regardless of graph settings. Only wedges that are selected will generate graphs. 3 Press the Print Preview button. 4 In the lower right corner of the print preview screen you can customize your report. Select or de-select Include Graphs, Include Assay Definition and Include Ct Values check-boxes. For larger segments, graphs can extend across many pages. 5 Click the Save-as button to save the report as an Adobe Acrobat or Microsoft Excel format. Click the Print button to generate a printout. Sincerely, Valerie Cimmarusti Vice President, Quality, Regulatory and Clinical Affairs

18 Simplexa HSV 1 & 2 Direct REF MOL2150 Rev. E A real-time PCR assay intended for the in vitro qualitative detection and differentiation of HSV-1 and/or HSV-2 viral DNA. For in vitro Diagnostic Use INTENDED USE The Focus Diagnostics Simplexa HSV 1 & 2 Direct assay is intended for use on the 3M Integrated Cycler instrument for the qualitative detection and differentiation of HSV-1 and HSV-2 present in cerebrospinal fluid (CSF) or genital swabs from patients with signs and symptoms of HSV-1 or HSV-2 infection of the central nervous system (CNS) or genitalia. This test is an aid in the differential diagnosis of HSV-1 and HSV-2 CNS and genital infections. Negative results do not preclude HSV-1 and/or HSV-2 virus and should not be used as sole basis for treatment or other patient management decisions. The assay is not intended for use as a screening test for the presence of HSV-1 and HSV-2 in blood or blood products. The assay is for professional use only. SUMMARY AND EXPLANATION HSV-1 and HSV-2 are members of the alpha-herpesviridae subfamily. HSV is an enveloped virus with a capsid containing viral DNA. Although HSV-1 and HSV-2 are closely related, the two viruses are serologically and genetically distinct. 1,2 Genital herpes caused by HSV is one of the most common sexually transmitted diseases. HSV primary infection or reactivation of latent disease can cause cutaneous or mucosal lesions. 1,3 These lesions are usually self-limited but severe disease can occur in compromised patients, pregnant woman and infants. The diagnosis of herpes lesions relies predominately on culture or direct fluorescent antibody (DFA) staining methods 3,4. However, these methods are labor intensive, time consuming, and subjective. 1,5 Rapid and accurate testing for HSV infection utilizing molecular detection can be critical for both therapeutic decision-making and infection control. 4,5 Encephalitis is inflammation of the brain associated with clinical evidence of neurologic dysfunction. Of the pathogens reported to cause encephalitis, the majority are viruses 6. In general, the most commonly identified etiologies in the United States are herpes simplex virus, West Nile virus, and the enteroviruses, followed by other herpesviruses. 6 HSV causes about 5 10% of all encephalitis cases, and is one of the most common causes of identified sporadic encephalitis globally. 6 HSV encephalitis occurs in all ages, and during all seasons. HSV-1 encephalitis is more common in adults; and HSV-2 encephalitis is more common in neonates. 6 One study reported a neonatal herpes rate of 1 case per 3,200 live births in the U.S. 7 Clinical features involved with HSV encephalitis include fever, hemicranial headache, language and behavioral abnormalities, memory impairment, and seizures. 6 The utility of nucleic acid amplification testing (e.g., PCR) of CSF specimens has greatly increased the ability to diagnose infections of the CNS, especially viral infections caused by the herpesviruses. 6 CSF cultures are generally of limited value in the determination of the viral causes of encephalitis but are very important in the diagnosis of bacterial and fungal infections. 6 Infectious Diseases Society of America (IDSA) recommends herpes simplex PCR testing on all CSF specimens in patients with encephalitis. For encephalitis patients with a negative herpes simplex PCR result, consideration should be given to repeating the test 3 7 days later for patients demonstrating a compatible clinical syndrome or temporal lobe localization on neuroimaging. 6 The American Congress of Obstetricians and Gynecologists (ACOG) has recognized that HSV PCR is times more sensitive than viral culture, that samples for PCR are easier to maintain, and that PCR is the test of choice in the diagnosis of infections of the central nervous system (encephalitis and meningitis). 8 PRINCIPLES OF THE PROCEDURE The Simplexa HSV 1 & 2 Direct assay system is a real-time PCR that enables the direct amplification, detection and differentiation of HSV-1 and/or HSV-2 DNA from unprocessed CSF and genital swab specimens without nucleic acid extraction. The system consists of the Simplexa HSV 1 & 2 Direct assay, the 3M Integrated Cycler (with 3M Integrated Cycler Studio Software), the Direct Amplification Disc and associated accessories.

19 Simplexa HSV 1 & 2 Direct kit Page 2 In the Simplexa HSV 1 & 2 Direct assay, bi-functional fluorescent probe-primers are used together with corresponding reverse primers to amplify HSV-1, HSV-2 and internal control targets. Well conserved regions of the HSV-1 and HSV-2 DNA polymerase genes are targeted to identify HSV-1 and HSV-2 DNA respectively in the specimen. An internal control is used to detect PCR failure and/or inhibition. MATERIALS PROVIDED The Focus Diagnostics Simplexa HSV 1 & 2 Direct assay contains sufficient reagents for 24 reactions. Upon receipt, store at -10 to -30 ºC (do not use a frost-free freezer). Each vial contains sufficient material for a single reaction. Use within 30 minutes of thawing. Kit Description Component Name REF EC SYMBOL ON LABEL Abbreviated Name Cap Color Number of Vials Reactions per Vial/Kit Volume per Vial Simplexa HSV 1 & 2 Direct Reaction Mix MOL2151 REAG A RM Brown 24 1/24 50 µl Kit Component Component Description Contents DNA polymerase, buffer, dntps, template DNA (Internal Control) dye-labeled fluorescent probe-primers specific for detection of HSV-1 and/or HSV-2 and for the DNA Internal Control. Target Probe Fluorophore (Dye) Excitation (nm) Emission (nm) Targeted Gene Simplexa HSV 1 & 2 Direct Reaction Mix (RM) HSV-1 CFR HSV-1 DNA polymerase HSV-2 FAM HSV-2 DNA polymerase DNA Internal Control Q NA Simplexa HSV 1 & 2 Kit Barcode Card Assay specific parameters. MATERIALS SUPPLIED SEPARATELY 1. Direct Amplification Disc Kit (REF MOL1455) a) Direct Amplification Discs for use on the 3M Integrated Cycler MATERIALS REQUIRED BUT NOT SUPPLIED 1. 3M Integrated Cycler with 3M Integrated Cycler Studio Software version 6.0 or higher 2. Simplexa HSV 1 & 2 Positive Control Pack (REF MOL2160) µl fixed volume pipette (VWR Signature Fixed Volume Ergonomic High-Performance Pipette Model VWR FE50 or equivalent) 4. Sterile, nuclease-free disposable pipette tips with filters (Extra Long tips 91 mm are recommended for pipetting directly from primary collection tubes). 5. Freezer (manual defrost) at -10 to -30 C (for kit component and specimen frozen storage) 6. Refrigerator at 2 to 8 C (for specimens) 7. Disposable, powder-free gloves RECOMMENDED MATERIALS 1. Synthetic CSF Golden West Biologicals Catalog number OH1030-S, SeraCare Catalog number HSP-515 or equivalent for use as a No Template Control (NTC) when testing CSF samples. 2. Universal Transport Media (UTM) to use as a No Template Control (NTC) when testing genital swab samples. REAGENT HANDLING AND STORAGE 1. Store reagents at -10 to -30 C (do not use a frost-free freezer). 2. Allow reagents to thaw at room temperature (approximate range 18 to 25 C) before use.

20 Simplexa HSV 1 & 2 Direct kit Page 3 3. Do not use kits or reagents beyond their expiration dates. 4. After removing Reaction Mix from freezer storage, initiate the test within 30 minutes. 5. Do not vortex the Reaction Mix. 6. Do not refreeze the Reaction Mix. WARNINGS AND PRECAUTIONS 1. Wear personal protective equipment, such as (but not limited to) gloves and lab coats when handling kit reagents. Wash hands thoroughly when finished performing the test. 2. Do not smoke, drink, eat, handle contact lenses or apply make-up in areas where kit reagents and/or human specimens are being used. 3. Dispose of unused kit reagents and human specimens according to local, state and federal regulations. 4. Treat all specimens and discs as capable of transmitting infectious agents. Contamination of patient specimens or reagents can produce erroneous results. Use good laboratory practices and control workflow. 9,10 5. Only use the protocol described in this insert. Deviations from the protocol or the use of times or temperatures other than those specified may give erroneous results. 6. Assay setup should be performed at room temperature (approximate range 18 to 25 C). 7. Use fixed volume pipettes or equivalent for sample and Reaction Mix. 8. Avoid touching the under side of the foil that will be in contact with the wells and disc surface. 9. To prevent potentially erroneous results, make sure that the sample is added to the Sample input well. 10. Finish loading and applying adhesive foil cover to one set of Sample and Reaction wells before opening the foil of adjacent set(s)s of Sample and Reaction wells. 11. Initiate the run within 30 minutes of removing the Reaction Mix vial from the freezer. 12. Do not attempt to remove adhesive foil cover from wedges that have been used or attempt to re-use Sample and Reaction ports that have been used in previous runs. 13. Discs may be reused until all 8 wedges have been used. Dispose of used discs without detaching foil cover in a biodazardous waste container. 14. After each use store DAD discs flat with the numbered foil side up. 15. Reaction Mix contains > 1% glycerol, which may cause irritation upon inhalation or skin contact. Upon inhalation or skin contact, first aid measures should be taken. 16. If kit packaging or contents appear to be broken or damaged do not use and contact Focus Diagnostics. Contact information is on the last page of this document. 17. The spectral matrix must be installed in each 3M Integrated Cycler and should not be changed unless an updated QR code for the instrument is provided by Focus Diagnostics. The spectral matrix is unique to each 3M Integrated Cycler. The spectral matrix was provided with the 3M Integrated Cycler instrument on the cover of the 3M Integrated Cycler Hardware Manual. If the matrix label will not scan or cannot be found contact Focus Diagnostics. The contact information is on the last page of this document. 18. Not installing or changing the spectral matrix can result in false results. INSTRUCTIONS FOR USE A. SPECIMEN COLLECTION CSF Sample Type Acceptable specimen type is CSF. Specimens should be transported on ice and stored at 2 to 8 C for up to 7 days post collection. If there is a greater than 7 days delay before processing of the specimen, store specimen at -70 C. Genital Swab Sample Type Acceptable specimen type is genital swabs stored in BD UVT, Remel M4, Remel M4RT, Remel M5, Remel M6 or UTM transport media. Follow the manufacturers package inserts for collection media and acceptable swab types. Do not use calcium alginate swabs, as they may contain substances that inhibit PCR testing. Specimens should be transported on ice and stored at 2 to 8 C for up to 7 days post collection. If there is a greater than 7 days delay before processing of the specimen, store specimen at -70 C. B. REAL-TIME PCR INSTRUMENT SETUP 1. Refer to the 3M Integrated Cycler Operator Manual for details on how to configure the 3M Integrated Cycler Studio Software to add an assay definition set up and analyze runs on the 3M Integrated Cycler. C. DIRECT AMPLIFICATION DISC LOADING AND REAL-TIME PCR AMPLIFICATION NOTE: No sample extraction is needed prior to PCR amplification step. 1. Select samples that need to be tested.

21 Simplexa HSV 1 & 2 Direct kit Page 4 2. Thaw Reaction Mix vials at room temperature (approximate range 18 to 25 C). Thaw one Reaction Mix vial for each sample or control to be tested. 3. Scan the barcode on the Simplexa HSV 1 & 2 Direct Reaction Mix vial or barcode card. 4. Scan the disc barcode on the Direct Amplification Disc (DAD). 5. Scan or type in each sample identifier. 6. For one wedge at a time, peel the adhesive foil back to expose the Sample (SAMPLE) and Reaction (R) wells without completely removing the adhesive foil cover (Figure 1 & 2). Avoid touching the under side of the foil that will be in contact with the wells and disc surface. 7. Ensure that the Reaction Mix is completely thawed. Briefly spin down the tubes as needed. (Do not vortex the Reaction Mix). 8. Use the fixed volume pipette to transfer 50 µl of the Reaction Mix into Reaction (R) well. 9. Use the fixed volume pipette to transfer 50 µl of sample or control; pipette sample or control into Sample well (SAMPLE). 10. Cover the wedge sealing the wells with the peeled adhesive foil, pressing down firmly near the edge of the wedge. If the original foil is torn do not load the wells in the wedge. Instead load another wedge. 11. Tear off the tab portion of the foil cover along the perforation. 12. Repeat steps 6 to 11 for the next sample(s). 13. Load the sealed DAD into the 3M Integrated Cycler and start the run. Figure 1 - Disc with pre-use foil lifted from Sample and Reaction Wells for wedge #3 Figure 2 Sample [Sample] and Reaction [R] Wells NOTES (for informational purposes - no user action/interpretation required): 1. Focus Diagnostics kits may contain version numbers for Assay Definitions. If the version number exists, it will be appended to the Assay Definition i.e. Sample IVD Assay.2. When multiple versions exist, the software automatically uses the assay definition associated with the scanned lot number. QUALITY CONTROL Simplexa TM HSV 1 & 2 Positive Control Pack (MOL2160) may be used as an external control for Quality Control (QC) testing, training or proficiency testing. Synthetic CSF is recommended as a No Template Control (NTC) when testing CSF samples and UTM is recommended as a NTC when testing genital swab samples. Quality control ranges have been established as indicated in the table below. If the controls are not within these parameters, patient results should be considered invalid and the assay repeated. Focus recommends testing controls once per day. Each laboratory should establish its own QC ranges and frequency of QC testing based on applicable local laws, regulations and standard good laboratory practice. Refer to the Simplexa TM HSV 1 & 2 Positive Control package insert (PI.MOL2160) for instructions on testing the positive control. Expected Control Control Type HSV-1 HSV-2 DNA Internal Control (DNA IC) Simplexa HSV 1 & 2 1 Positive Control Detected Detected Not applicable2 No Template Control (NTC) Not Detected Not Detected Valid 1 Typical Ct values for the Positive Control range between 25 to Detection of the Simplexa DNA Internal Control (DNA IC) is not required for a valid result when HSV is detected.

22 Simplexa HSV 1 & 2 Direct kit Page 5 RESULTS Upon completion of the run, the software automatically calculates and displays results. 1. For each accession ID (Sample ID) entered, the software displays a result ( Detected, Not Detected, Invalid or EC500 ) for HSV-1 and HSV-2. a. Detected result points to the presence of HSV-1 and/or HSV-2 DNA in the patient sample. b. Not Detected result points to the absence of HSV-1 and/or HSV-2 DNA in the patient sample. c. Invalid result points to the inability to determine presence or absence of HSV-1 and/or HSV-2 DNA in the patient sample. This result may be due to 1) DNA Internal Control (DNA IC) failure, or 2) failure to detect sufficient specimen. The sample needs to be re-tested. See Invalid section below. d. EC500 result points to a data quality error for the particular viral analyte(s). The software was unable to determine a valid amplification for that analyte(s). The sample should be re-tested. e. EC505 result indicates a data quality error for the viral analyte. The software was unable to determine a valid amplification for the analyte. Contact Technical Assistance at Focus Diagnostics which can be found on the last page of this document. 2. Print the report as needed. a. Export the results as needed INVALID RESULTS In case of an Invalid result, re-test the sample with a new Reaction Mix vial from the same kit or a new kit. If the problem is unresolved, contact Focus Diagnostics Technical Services department. LIMITATIONS 1. For in vitro diagnostic use. 2. For Export Use Only. 3. from this test must be considered in conjunction with the clinical history, epidemiological data and other laboratory information available to the clinician evaluating the patient. 4. The detection of viral nucleic acid is dependent upon proper sample collection, transport, handling and storage. Failure to observe proper procedures in any one of these steps can lead to incorrect results. 5. The prevalence of viral infections may affect the test s predictive value. 6. Negative results do not rule out HSV infections of the CNS and should not be used as the sole basis for treatment or other patient management decisions. 7. False-negative results may occur if the viruses are present at a level that is below the analytical sensitivity of the assay or if the virus has genomic mutations, insertions, deletions, or rearrangements or if performed very early in the course of illness. 8. For encephalitis patients with a negative herpes simplex PCR result, consideration should be given to repeating the test 3 7 days later for patients demonstrating a compatible clinical syndrome or temporal lobe localization on neuroimaging As with other tests, false-positive results may occur. Repeat testing or testing with a different device may be indicated in some settings. 10. A positive result by this test cannot rule out infections caused by other viral or bacterial pathogens. Viral nucleic acids may persist in vivo independent of virus viability. Detection of target analyte(s) does not imply that the corresponding viruses are infectious or are the causative agent for clinical symptoms. 11. When very high levels of HSV-1 are present with very low levels of HSV-2, the signal from the HSV-2 reaction may not be adequate to be detected, due to competitive interference. 12. The prevalence of viral infections may affect the test s predictive value. 13. This test is a qualitative test and does not provide the quantitative value of detected virus present. 14. The performance of this test has not been established for screening of blood or blood products for the presence of HSV or for use with samples other than CSF or genital swabs. 15. The performance of this test has not been established for immunocompromised individuals. 16. The performance of this test has not been established for monitoring treatment of HSV infection of the CNS. 17. Information on the Simplexa HSV 1 & 2 Direct Reaction Mix vial can only be transferred into the 3M Integrated Cycler Studio through a bar-code scanner. If the scanner is not working, or if you are unable to transfer the information for any reason, contact Focus Diagnostics Technical Services.

23 Simplexa HSV 1 & 2 Direct kit Page 6 PERFORMANCE CHARACTERISTICS EXPECTED VALUES CSF Sample Type The observed expected values of HSV-1 and HSV-2 in the prospective population of the Simplexa HSV 1 & 2 Direct assay s clinical study varied between institutions and are shown in the table below. Sample Population Prospective Sample Collection Site No. of Samples Expected Values Based on Simplexa HSV 1 & 2 Direct Assay HSV-1 HSV % (0/31) 6.5% (2/31) % (1/24) 0.0% (0/24) % (4/94) 5.3% (5/94) % (0/15) 0.0% (0/15) All % (5/164) 4.3% (7/164) Not Detected 93.5% (29/31) 95.8% (23/24) 90.4% (85/94) (15/15) 92.7% (152/164) Age Category HSV -1 HSV -2 Prospective - Number of Samples by Simplexa HSV 1 & 2 Direct Assay Combined Female Male Not Detected All HSV- 1 HSV- 2 Not Detected All HSV- 1 HSV- 2 Not Detected From Birth to 1 month of age >1 month to 2 years of age >2 years to 12 years of age >12 years to 21 years of age >21 years to 60 years of age >60 years of age All All

24 Simplexa HSV 1 & 2 Direct kit Page 7 EXPECTED VALUES Genital Swab Sample Type The observed expected values of HSV-1 and HSV-2 in the prospective population of the Simplexa HSV 1 & 2 Direct assay s clinical study varied between institutions and are shown in the table below. ELVIS Culture Result (Prevalence) Simplexa Result (Expected Values) Sequencing Result (Expected Values) Sample Collection Site ID All HSV 1&2 Not Detected HSV-1 Detected, HSV-2 Not Detected HSV-1 Not Tested & HSV-2 Detected Not- Evaluable HSV 1&2 Detected HSV 1 &2 Not Detected HSV-1 Detected & HSV-2 Not Detected HSV-1 Not Detected & HSV-2 Detected Not- Evaluable HSV-1 Detected HSV 1&2 Not Detected HSV-2 Detected Not- Evaluable % (165/256) 14.1% (36/256) 20.3% (52/256) 1.2% (3/256) 0.4% (1/256) 53.9% (138/256) 16.0% (41/256) 28.5% (73/256) 1.2% (3/256) 16.0% (41/256) 54.3% (139/256) 27.3% (70/256) 2.3% (6/256) % (58/74) 6.8% (5/74) 6.8% (5/74) 8.1% (6/74) 1.4% (1/74) 73.0% (54/74) 9.5% (7/74) 12.2% (9/74) 4.1% (3/74) 6.8% (5/74) 74.3% (55/74) 9.5% (7/74) 9.5% (7/74) % (46/78) 6.4% (5/78) 32.1% (25/78) 2.6% (2/78) 1.3% (1/78) 51.3% (40/78) 19.2% (15/78) 26.9% (21/78) 1.3% (1/78) 16.7% (13/78) 51.3% (40/78) 28.2% (22/78) 3.8% (3/78) % (72/140) 12.1% (17/140) 24.3% (34/140) 12.1% (17/140) 1.4% (2/140) 47.1% (66/140) 20.0% (28/140) 27.1% (38/140) 4.3% (6/140) 18.6% (26/140) 47.1% (66/140) 28.6% (40/140) 5.7% (8/140) % (52/89) 11.2% (10/89) 29.2% (26/89) 1.1% (1/89) 0.0% (/89) 51.7% (46/89) 21.3% (19/89) 25.8% (23/89) 1.1% (1/89) 22.5% (20/89) 49.4% (44/89) 25.8% (23/89) 2.2% (2/89) % (52/81) 6.2% (5/81) 23.5% (19/81) 6.2% (5/81) 0.0% (/81) 56.8% (46/81) 16.0% (13/81) 23.5% (19/81) 3.7% (3/81) 17.3% (14/81) 48.1% (39/81) 22.2% (18/81) 12.3% (10/81) All % (445/718) 10.9% (78/718) 22.4% (161/718) 4.7% (34/718) 0.7% (5/718) 54.3% (390/718) 17.1% (123/718) 25.5% (183/718) 2.4% (17/718) 16.6% (119/718) 53.3% (383/718) 25.1% (180/718) 5.0% (36/718)

25 Simplexa HSV 1 & 2 Direct kit Page 8 Gender Age Group All Female Male Sample Demographics by Simplexa HSV 1 & 2 Direct Assay HSV 1&2 Detected Simplexa HSV 1 & 2 Direct Result HSV 1&2 Not Detected HSV1 Detected & HSV2 Not Detected HSV1 Not Detected & HSV2 Detected Not-Evaluable More than 1 month to 2 Years of age 2 0.0% (0/2) 50.0% (1/2) 50.0% (1/2) 0.0% (0/2) 0.0% (0/2) More than 2 Years of age to 12 Years of age More than 12 Years of age to 18 Years of age More than 18 Years of age to 21 Years of age % (0/10) 90.0% (9/10) 0.0% (0/10) 10.0% (1/10) 0.0% (0/10) % (0/56) % (0/61) More than 21 Years of age % (4/503) All % (4/632) 62.5% (35/56) 29.5% (18/61) 54.3% (273/503) 53.2% (336/632) 10.7% (6/56) 32.8% (20/61) 16.9% (85/503) 17.7% (112/632) 25.0% (14/56) 34.4% (21/61) 25.8% (130/503) 26.3% (166/632) 1.8% (1/56) 3.3% (2/61) 2.2% (11/503) 2.2% (14/632) More than 1 month to 2 Years of age 1 0.0% (0/1) 0.0% (0/1) (1/1) 0.0% (0/1) 0.0% (0/1) More than 2 Years of age to 12 Years of age More than 12 Years of age to 18 Years of age More than 18 Years of age to 21 Years of age More than 21 Years of age % (1/66) All % (1/84) 2 0.0% (0/2) (2/2) 0.0% (0/2) 0.0% (0/2) 0.0% (0/2) 3 0.0% (0/3) (3/3) 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) % (0/12) 33.3% (4/12) 25.0% (3/12) 41.7% (5/12) 0.0% (0/12) 68.2% (45/66) 64.3% (54/84) 10.6% (7/66) 13.1% (11/84) 18.2% (12/66) 20.2% (17/84) 1.5% (1/66) 1.2% (1/84) Unknown Unknown 2 0.0% (0/2) 0.0% (0/2) 0.0% (0/2) 0.0% (0/2) (2/2) All % (5/718) 54.3% (390/718) 17.1% (123/718) 25.5% (183/718) 2.4% (17/718)

26 Simplexa HSV 1 & 2 Direct kit Page 9 CLINICAL AGREEMENT CSF Sample Type A total of 219 CSF samples were prospectively or retrospectively collected from patients with signs and symptoms of herpes simplex virus (HSV) central nervous system (CNS) infection by eight external sites. All sites sent the frozen samples to Focus Diagnostics. Two aliquots were prepared from each sample and the samples were then blinded for testing. One aliquot was sent to an external testing site while the other aliquot was retained at Focus Diagnostics for sequencing. Five external sites performed testing with the Simplexa HSV 1 & 2 Direct assay which included four of the collection sites. Focus Diagnostics performed testing on the samples using PCR followed by analysis with two bi-directional sequencing assays. from the Simplexa HSV 1 & 2 Direct assay were compared to the results from two bi-directional sequencing assays (comparator). Neural imaging/clinical impression results documented in a case report form (CRF) as determined by the patient s attending physician and other clinical information such as chemistries, bacterial culture, MRI/CT scans and in-house PCR results were collected for all patients. The Final Diagnosis takes into consideration all of the laboratory findings and the results of a PCR test performed locally along with clinical presentation of the patient. No dual positive (both HSV-1 Positive and HSV-2 Positive) samples were found. The table below shows the distribution of the different clinical parameters collected from patients who were confirmed positive and negative by the comparator. Parameter Patients which are positive by the comparator for HSV (Number and %) Patients which are negative by the comparator for HSV (Number and %) MRI results suggestive of HSV infection 18/65 (27.7%) 9/154 (5.8%) MRI results not suggestive of HSV infection 43/65 (66.2%) 121/154 (78.6%) MRI results not available 4/65 (6.2%) 24/154 (15.6%) Final Diagnosis positive for HSV infection 61/65 (93.8%) 33/154 (21.4%) Final Diagnosis negative for HSV infection 4/65 (6.2%) 121/154 (78.6%) Bacteria culture positive 2/65 (3.1%) 4/154 (2.6%) Bacteria culture negative 63/65 (96.9%) 145/154 (94.2%) Bacteria culture not performed 0/65 (0.0%) 5/154 (3.2%) The following tables summarize the results of the Simplexa HSV 1 & 2 Direct assay s agreement with the comparator (results of two sequencing PCR assays). Three hundred (300) base-pair dideoxy DNA sequencing assays were validated for two different HSV gene targets. SYBR Green PCR using extracted patient sample DNA was used to test all samples. Amplicons from positive samples were used for bidirectional sequencing. Sequence requirements had a phred quality score 20 (for each base) and a continuous read length 200 and a 2x coverage 180. Sequence alignments were analyzed against the GenBank database using the BLAST search program to determine if the patient sample was positive for HSV-1 or HSV-2. Simplexa HSV-1: Prospective Samples HSV-2: Prospective Samples Comparator: Two PCR/SEQ Simplexa Comparator: Two PCR/SEQ Detected Not Detected Total Detected Not Detected Total Detected 3 2* 5 Detected 6 1 ## 7 Not Detected Not Detected 1 # Total Total PPA (3/3) 95% CI: 43.8 to PPA 85.7%(6/7) 95% CI: 48.7 to 97.4% NPA 98.8%(159/161) 95% CI: 95.6 to 99.7% NPA 99.4%(156/157) 95% CI: 96.5 to 99.9% *1/2 sample had a final diagnosis of meningitis from the chart information. # Sample was collected from patient with a final diagnosis of meningitis from the chart information. ## Sample was collected from patient with a final diagnosis of meningitis from the chart information. Note: PPA: Positive Percent ; NPA: Negative Percent

27 Simplexa HSV 1 & 2 Direct kit Page 10 Fifty five (55) retrospective/preselected HSV positive (as determined by the collection sites) samples from four sites were collected between 2004 and 2013 and confirmed positive by the comparator were used to supplement the prospective study to evaluate the sensitivity of the Simplexa HSV 1 & 2 Direct. The results are shown in the table below. Retrospective/ Preselected Positive Samples Positive Percent (PPA) Simplexa Comparator: Two PCR/SEQ HSV-1 HSV-2 HSV HSV Not Detected 0 0 Total PPA HSV-1 (13/13) 95% CI: 77.2 to HSV-2 (42/42) 95% CI: 91.6 to CLINICAL AGREEMENT Genital Swab Sample Type A total of 718 genital swab samples were prospectively collected from patients with signs and symptoms of genital herpes simplex virus (HSV) by 6 geographically diverse locations. All sites collected and tested the genital swab samples and sent samples to a central lab for culture testing. For culture, each sample was tested for HSV- 2 first and if positive for HSV-2 no further testing was performed. Samples that were HSV-2 culture negative were further tested for HSV-1 culture positivity.. Dual positives were not identified in the assay. The retained samples were sent to Focus Diagnostics and tested in a validated bi-directional sequencing assay and dual positives were detected. HSV-1 Bi-Directional Sequencing Clinical - (Overall for HSV-1) Simplexa HSV 1 & 2 Direct Validated Sequencing Method Detected Not Detected Total Detected a 125 Not Detected 3 b Total %PPA 97.4%(114/117) 95% CI: 92.7% to 99.1% %PPV 91.2%(114/125) 95% CI: 84.9% to 95.0% %NPA 98.0%(546/557) 95% CI: 96.5% to 98.9% %NPV 99.5%(546/549) 95% CI: 98.4% to 99.8% a Of the eleven (11) discordant samples, two (2) samples were Detected as HSV-1, six (6) were Not Detected for both HSV-1 and HSV-2 and three (3) samples were Detected as HSV-2 and not tested for HSV-1 when tested using the culture method. b Of the three (3) discordant samples, two (2) were Not Detected for both HSV-1 and HSV-2 and one (1) sample was Detected as HSV-2 when tested using the culture method.

28 Simplexa HSV 1 & 2 Direct kit Page 11 HSV-2 Bi-Directional Sequencing Clinical - (Overall for HSV-2) Simplexa Validated Sequencing Method Detected Not Detected Total Detected c 185 Not Detected 3 d Total %PPA 98.3%(174/177) 95% CI: 95.1% to 99.4% %PPV 94.1%(174/185) 95% CI: 89.7% to 96.6% %NPA 97.8%(486/497) 95% CI: 96.1% to 98.8% %NPV 99.4%(486/489) 95% CI: 98.2% to 99.8% c Of the eleven (11) discordant samples, one (1) sample was Detected as HSV-2 and not tested for HSV-1 and ten (10) samples were Not Detected for both HSV-1 and HSV-2 when tested using the culture method. d Of the three (3) discordant samples, three (3) samples were Not Detected for both HSV-1 and HSV-2 when tested using the culture assay method. REPRODUCIBILITY - CSF Sample Type Reproducibility for the Simplexa HSV 1 & 2 Direct assay was evaluated. Three investigative sites assessed the device's intersite, inter-day and inter/intra-assay reproducibility. Each of the laboratories tested positive control and a panel of five contrived sample pools including a low (approximately 1 times LoD) and medium positive (approximately 4 times LoD) for each analyte and a high negative. The high negative sample contained a small amount of HSV-1 and HSV-2, and it was designed to be negative approximately 95% of the time. The assays were performed in triplicate on five different days. Each site had two operators who each assayed the entire sample panel and positive control once per day, for a total of two sets of data per day. Combined results for all sites are presented in the tables below. HSV-1 Result Sample HSV-1 Low Positive HSV-1 Medium Positive HSV-2 Low Positive HSV-2 Medium Positive High Negative Positive Control Total % with Expected Site 1 Site 2 Site 3 Avg. Ct Total %CV a NA NA a NA NA a NA NA % with Expected Avg. Ct Total %CV % with Expected Avg. Ct Total %CV Total % with Expected (90/90) (90/90) 95% CI 95.9% to 95.9% to 95.9% a NA NA a NA NA (90/90) a to 95.9% a NA NA a NA NA (90/90) a to 95.9% a NA NA a NA NA (90/90) a to (180/180) (180/180) (180/180) (90/90) (540/540) 95.9% to 99.3% to

29 Simplexa HSV 1 & 2 Direct kit Page 12 a) Expected of HSV-2 Low Positive, HSV-2 Medium Positive and High Negative samples are Negative for HSV-1. HSV-2 Result Sample HSV-1 Low Positive HSV-1 Medium Positive HSV-2 Low Positive HSV-2 Medium Positive High Negative Positive Control Total % with Expected Site 1 Site 2 Site 3 Avg. Ct Total %CV % with Expected Avg. Ct Total %CV % with Expected Avg. Ct Total %CV Total % with Expected 95% CI 95.9% b NA NA b NA NA b NA NA (90/90) b to 95.9% b NA NA b NA NA b NA NA (90/90) b to 96.7% (29/30) % (27/30) % (25/30) % (81/90) (90/90) 82.1% to 94.6% 95.9% to 89.1% 93.3% (28/30) b % (29/30) b 38.4 NA 96.7% (29/30) b 39.1 NA 95.6% (86/90) b to 98.3% % (177/180) 97.8% (176/180) 96.7% (174/180) (90/90) 97.6% (527/540) b) Expected of HSV-1 Low Positive, HSV-1 Medium Positive and High Negative samples are Negative for HSV % to 95.9% to 98.6% DNA IC Result Sample HSV-1 Low Positive HSV-1 Medium Positive HSV-2 Low Positive HSV-2 Medium Positive High Negative Positive Control % with Expected Site 1 Site 2 Site 3 Avg. Ct Total %CV % with Expected Avg. Ct Total %CV % with Expected Avg. Ct Total %CV Total % with Expected (90/90) (90/90) (90/90) (90/90) (90/90) (90/90) 95% CI 95.9% to 95.9% to 95.9% to 95.9% to 95.9% to 95.9% to Total (180/180) (180/180) (180/180) (540/540) 99.3% to