Who is the manufacturer of cobas HSV 1 and 2 Test?

Roche Molecular Systems, Inc. filed the 510(k) submission for cobas HSV 1 and 2 Test (K150617).

What is the FDA product code for cobas HSV 1 and 2 Test?

OQO. It is classified under 21 CFR 866.3305 as a Class 2 device in the Microbiology panel.

What is the regulatory pathway for cobas HSV 1 and 2 Test?

510(k) clearance (submission type: Traditional).

Who can help prepare an FDA 510(k) submission like cobas HSV 1 and 2 Test?

Innolitics — a medical-device software consultancy — provides regulatory and engineering support for FDA 510(k) submissions. See https://innolitics.com/services/510ks/ or contact https://innolitics.com/contact.

cobas HSV 1 and 2 Test

K150617 · Roche Molecular Systems, Inc. · OQO · Jun 1, 2015 · Microbiology

Device Facts

Record ID	K150617
Device Name	cobas HSV 1 and 2 Test
Applicant	Roche Molecular Systems, Inc.
Product Code	OQO · Microbiology
Decision Date	Jun 1, 2015
Decision	SESE
Submission Type	Traditional
Regulation	21 CFR 866.3305
Device Class	Class 2

Intended Use

The cobas® HSV 1 and 2 Test on the cobas® 4800 system is an automated, qualitative in vitro diagnostic test, that utilizes real-time polymerase chain reaction (PCR), for the direct detection and differentiation of Herpes simplex virus 1 and 2 (HSV-1 and HSV-2) DNA in clinician-collected, external anogenital lesion specimens from symptomatic male and female patients. The cobas® HSV 1 and 2 Test is intended for use as an aid in diagnosis of anogenital HSV-1 and HSV-2 infections in symptomatic patients. Warning: The cobas® HSV 1 and 2 Test is not FDA cleared for use with cerebrospinal fluid (CSF) and is not intended to be used for prenatal screening or for individuals under the age of 18 years.

Device Story

The cobas® HSV 1 and 2 Test is an automated, qualitative in vitro diagnostic assay for the detection and differentiation of HSV-1 and HSV-2 DNA. It uses clinician-collected external anogenital lesion swabs in MSwab medium as input. The device performs automated sample preparation (lysis, nucleic acid extraction using magnetic glass particles) and real-time PCR amplification on the cobas® 4800 system. It targets conserved regions of HSV-1 (Thymidine Kinase, DNA Polymerase) and HSV-2 (Thymidine Kinase, Glycoprotein B) genes. Fluorescent-labeled TaqMan® probes detect amplicons in real-time. An internal control (lambda phage DNA) monitors lysis and PCR inhibition. The system outputs qualitative results for HSV-1 and HSV-2. It is used in clinical settings to aid healthcare providers in diagnosing anogenital HSV infections in symptomatic patients, facilitating clinical decision-making regarding patient management.

Clinical Evidence

Prospective multi-site study of 408 specimens compared the device to a composite reference method (culture and PCR/sequencing). HSV-1 sensitivity 92.9% (95% CI: 85.3-96.7%), specificity 98.8% (95% CI: 96.9-99.5%). HSV-2 sensitivity 97.0% (95% CI: 93.2-98.7%), specificity 94.6% (95% CI: 91.0-96.8%).

Technological Characteristics

Real-time PCR assay using TaqMan® probes. Automated nucleic acid extraction via magnetic glass particles. Targets: HSV-1 Thymidine Kinase/DNA Polymerase and HSV-2 Thymidine Kinase/Glycoprotein B. Platform: cobas® 4800 system (cobas x 480 for extraction, cobas z 480 for amplification/detection).

Indications for Use

Indicated for symptomatic male and female patients aged 18 years or older presenting with external anogenital lesions to aid in the diagnosis of HSV-1 and HSV-2 infections. Contraindicated for use with cerebrospinal fluid (CSF), prenatal screening, or individuals under 18 years of age.

Regulatory Classification

Identification

Herpes simplex virus serological assays are devices that consist of antigens and antisera used in various serological tests to identify antibodies to herpes simplex virus in serum. Additionally, some of the assays consist of herpes simplex virus antisera conjugated with a fluorescent dye (immunofluorescent assays) used to identify herpes simplex virus directly from clinical specimens or tissue culture isolates derived from clinical specimens. The identification aids in the diagnosis of diseases caused by herpes simplex viruses and provides epidemiological information on these diseases. Herpes simplex viral infections range from common and mild lesions of the skin and mucous membranes to a severe form of encephalitis (inflammation of the brain). Neonatal herpes virus infections range from a mild infection to a severe generalized disease with a fatal outcome.

Special Controls

*Classification.* Class II (special controls). The device is classified as class II (special controls). The special control for the device is FDA's revised guidance document entitled “Class II Special Controls Guidance Document: Herpes Simplex Virus Types 1 and 2 Serological Assays.” For availability of the guidance revised document, see § 866.1(e).

Predicate Devices

BD ProbeTec™ Herpes Simplex Viruses (HSV 1 & 2) Q⁺ Amplified DNA Assays (K103798)

Reference Devices

ELVIS® HSV ID/Typing Test System (K971662)

Related Devices

K180559 — HSV 1 & 2 ELITe MGB Kit; ELITe InGenius · Elitechgroup · Oct 29, 2018
K142156 — SEEGENE ANYPLEX II HSV-1/2 ASSAY · Seegene · Feb 13, 2015

Submission Summary (Full Text)

{0}------------------------------------------------ Image /page/0/Picture/1 description: The image shows the logo for the U.S. Department of Health & Human Services. The logo consists of a circular seal with the text "DEPARTMENT OF HEALTH & HUMAN SERVICES - USA" around the perimeter. Inside the circle is a stylized image of three human profiles facing to the right, with flowing lines representing hair or a cape. Food and Drug Administration 10903 New Hampshire Avenue Document Control Center - WO66-G609 Silver Spring, MD 20993-0002 June 1, 2015 ROCHE MOLECULAR SYSTEMS, INC. DAVID W. GATES, PH.D. SENIOR DIRECTOR, REGULATORY AFFAIRS 4300 HACIENDA DRIVE PLEASANTON, CA 94588-2722 Re: K150617 Trade/Device Name: cobas® HSV 1 and 2 Test Regulation Number: 21 CFR 866.3305 Regulation Name: Herpes Simplex Virus Nucleic Acid Amplification Assay Regulatory Class: II Product Code: 000 Dated: March 9, 2015 Received: March 10, 2015 Dear Dr. Gates: We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food. Drug. and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading. If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21. Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register. Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801); medical device reporting (reporting of medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820); and if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050. {1}------------------------------------------------ If you desire specific advice for your device on our labeling regulation (21 CFR Part 801), please contact the Division of Industry and Consumer Education at its toll-free number (800) 638-2041 or (301) 796-7100 or at its Internet address http://www.fda.gov/MedicalDevices/Resourcesfor You/Industry/default.htm. Also, please note the regulation entitled. "Misbranding by reference to premarket notification" (21CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance. You may obtain other general information on your responsibilities under the Act from the Division of Industry and Consumer Education at its toll-free number (800) 638-2041 or (301) 796-7100 or at its Internet address http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm. Sincerely yours, # Stephen J. Lovell -S for Sally A. Hojvat, M. Sc., Ph.D. Director Division of Microbiology Devices Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health Enclosure {2}------------------------------------------------ # Indications for Use 510(k) Number (if known) K150617 Device Name cobas® HSV 1 and 2 Test #### Indications for Use (Describe) The cobas® HSV 1 and 2 Test on the cobas® 4800 system is an automated, qualitative in vitro diagnostic test, that utilizes real-time polymerase chain reaction (PCR), for the direct detection and differentiation of Herpes simplex virus 1 and 2 (HSV-1 and HSV-2) DNA in clinician-collected, external anogenital lesion specimens from symptomatic male and female patients. The cobas® HSV 1 and 2 Test is intended for use as an aid in diagnosis of anogenital HSV-1 and HSV-2 infections in symptomatic patients. Box warning: Warning: The cobas® HSV 1 and 2 Test is not FDA cleared for use with cerebrospinal fluid (CSF) and is not intended to be used for prenatal screening or for individuals under the age of 18 years. | | | | Type of Use (Select one or both, as applicable) | |--|--|--|-------------------------------------------------| |--|--|--|-------------------------------------------------| X Prescription Use (Part 21 CFR 801 Subpart D) Over-The-Counter Use (21 CFR 801 Subpart C) #### CONTINUE ON A SEPARATE PAGE IF NEEDED. This section applies only to requirements of the Paperwork Reduction Act of 1995. #### *DO NOT SEND YOUR COMPLETED FORM TO THE PRA STAFF EMAIL ADDRESS BELOW.* The burden time for this collection of information is estimated to average 79 hours per response, including the time to review instructions, search existing data sources, gather and maintain the data needed and complete and review the collection of information. Send comments regarding this burden estimate or any other aspect of this information collection, including suggestions for reducing this burden, to: > Department of Health and Human Services Food and Drug Administration Office of Chief Information Officer Paperwork Reduction Act (PRA) Staff PRAStaff(@fda.hhs.gov "An agency may not conduct or sponsor, and a person is not required to respond to, a collection of information unless it displays a currently valid OMB number." {3}------------------------------------------------ # cobas® HSV 1 and 2 Test 510(k) Summary | Submitter Name | Roche Molecular Systems, Inc. | |-------------------------------|---------------------------------------------------------------------------------------| | Address | 4300 Hacienda Drive Pleasanton, CA 94588-2722 | | Contact | David Gates Phone: 925.730-8237 | | Date Prepared | February 27, 2015 | | Proprietary Name | cobas® HSV 1 and 2 Test | | Common Name | HSV 1 and 2 Test | | Regulation | 21 CFR 866.3305 | | Classification | Class II | | Product Code | OQO: Herpes Simplex Virus Nucleic Acid Amplification Assay | | Predicate Devices | BD ProbeTecTM Herpes Simplex Viruses (HSV 1 & 2) Qx Amplified DNA Assays (K103798) | | Establishment Registration | Branchburg: 2243471 Pleasanton: 3004141078 Indianapolis: 1823260 | {4}------------------------------------------------ #### 1. DEVICE DESCRIPTION The Roche Molecular Systems (RMS) cobas® HSV 1 and 2 Test utilizes real-time polymerase chain reaction (PCR) for detection of HSV-1 and HSV-2 DNA in clinician-collected external anogenital lesion specimens, collected in MSwab medium from symptomatic patients. The cobas® HSV 1 and HSV 2 Test contains two major processes: (1) automated sample preparation to extract nucleic acids from swab specimens; (2) PCR amplification of target DNA sequences using HSV-1 and HSV-2 specific primers, and real-time detection of cleaved fluorescent-labeled HSV-1 and HSV-2 specific oligonucleotide detection probes. An Internal Control (IC), containing unrelated randomized DNA sequence, is added to all samples prior to automated sample preparation and is amplified and detected simultaneously with each sample to monitor the entire process. The MSwab Collection. Transport and Preservation System (Copan Flock Technologies) is used for specimen collection, transportation and storage of specimen for the cobas " HSV 1 and HSV 2 Test. The cobas® HSV 1 and HSV 2 Test utilizes six reagent kits: - cobas® 4800 HSV 1 and HSV 2 Amplification/Detection Kit 1) - cobas® 4800 HSV 1 and HSV 2 Controls and Cofactor Kit 2) - cobas® 4800 System Wash Buffer Kit 3) - cobas® 4800 System Lysis Kit 1 4) - cobas® 4800 System Internal Control Kit 1 5) - cobas® 4800 System Sample Preparation Kit 6) {5}------------------------------------------------ ### Test Principle Target Selection: The cobas® HSV 1and 2 Test utilizes real-time PCR technology to detect the conserved regions of HSV-1 Thymidine Kinase and HSV-1 DNA Polymerase as well as HSV-2 Thymidine Kinase and HSV-2 Glycoprotein B genes. Fluorogenic target-specific probes are used for the detection of the amplified HSV-1 and HSV-2 DNA as well as Internal Control. Since two HSV type targets are detected with different fluorescent dyes, the cobas® HSV 1 and 2 Test has the ability to simultaneously detect and differentiate HSV-1 and HSV-2. Primer and probe oligonucleotide sequences were designed to select HSV-1 and HSV-2 conserved sequences without cross reacting to other viruses, or other bacterial organisms commonly found in human genital areas. Sample Preparation: Sample preparation for the cobas® HSV 1 and 2 Test is automated with the use of the cobas x 480 instrument. Organisms from swab specimens collected in MSwab medium are lysed with chaotropic agent, proteinase K, and SDS reagents. Released nucleic acids, along with added Internal Control DNA, are bound by magnetic glass particles. They are washed and then eluted into a small volume of buffer. The instrument then takes an aliquot of the eluted material and sets up the PCR reaction with an activated Master Mix. PCR Amplification and TaqMan® Detection: The PCR cycling steps and detection of target signal occurs in the cobas 2 480 Analyzer. The Master Mix reagent contains primer pairs and probes for HSV-1, HSV-2 and IC targets. If the target nucleic acid sequences are present, amplification with the corresponding primers will occur by a thermostable DNA polymerase, generating PCR products (amplicons). These products are detected by specific TaqMan probes containing a fluorescent dye and a quencher. Normally, the quencher suppresses the fluorescence of the dye. However, if the PCR product is present, the probe hybridizes to the product and gets cleaved by the 5' to 3' nuclease activity of the polymerase. This reaction allows the fluorescence to be emitted from the dye, and the signal is recorded in real time during each PCR cycle by the cobas 2 480 analyzer. The signal is interpreted by the cobas® 4800 System Software and reported as final results. {6}------------------------------------------------ ### Assay Controls RMS provides Negative Control (NC), Positive Control (PC) and Internal Control (IC) as reagent components in the cobas® HSV 1 and 2 Test - NC contains a buffer solution. The NC is processed in each run that contains a batch . of HSV specimens and should invalidate the run if there is contamination during the assay process that results in a positive signal in any of the assay target detection channels and/or if internal control signal is negative or invalid. - PC contains one plasmid that contains the HSV-1 target sequence, and a second plasmid that contains the HSV-2 target sequence, in the same buffer as NC. One PC is processed in each run that contains a batch of HSV specimens. The PC monitors the overall reagent and process integrity and will invalidate the run if results are outside of allowable ranges in the assay target detection channels and/or internal control signal is negative or invalid. - IC contains recombinant lambda phage with a generic DNA sequence, which is . amplified and detected along with HSV-1 and 2 targets but uses specific primers and probe that are different from the HSV-1 and 2 sequences. Since the IC DNA is contained in the phage capsule, it acts both as a specimen lysis control and as a control for PCR inhibition. The IC is added to all specimens, PC and NC in a run. ## cobas® 4800 System Description The cobas® 4800 System is a diagnostic system designed for sample preparation and real time amplification and detection of nucleic acid targets from clinical samples. The system hardware is unchanged from that originally approved for IVD use in PMA P100020 (cobas® HPV Test, April 19, 2011). The software version has been updated to software release 2.1 in order to support the expanded test menu. The updated software was cleared for the current tests on the cobas® 4800 system per Special 510(k) (K140887). {7}------------------------------------------------ #### 2. INTENDED USE The cobas® HSV 1 and 2 Test on the cobas® 4800 system is an automated, qualitative in vitro diagnostic test, that utilizes real-time polymerase chain reaction (PCR), for the direct detection and differentiation of Herpes simplex virus 1 and 2 (HSV-1 and HSV-2) DNA in cliniciancollected, external anogenital lesion specimens from symptomatic male and female patients. The cobas® HSV 1 and 2 Test is intended for use as an aid in diagnosis of anogenital HSV-1 and HSV-2 infections in symptomatic patients. Warning: The cobas® HSV 1 and 2 Test is not FDA cleared for use with cerebrospinal fluid (CSF) and is not intended to be used for prenatal screening or for individuals under the age of 18 years. #### 3. TECHNOLOGICAL CHARACTERISTICS The RMS cobas® HSV 1 and 2 Test is substantially equivalent in terms of its technological characteristics to the currently legally marketed predicate device, the BD ProbeTec "" Herpes Simplex Viruses (HSV 1 & 2) Q* Amplified DNA Assays on the BD Viper System in Extracted Mode, (K103798). Differences reside in the amplification technology. Although the candidate test utilizes real-time PCR while the predicate uses strand displacement, both methods are using the same basic principle to amplify low copies of nucleic acids to amounts which can subsequently be detected. | Similarities | | | |------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| | Characteristic | BD ProbeTec™ Herpes Simplex Viruses (HSV 1 & 2) Qx Amplified DNA Assays on the BD Viper System in Extracted Mode, (K103798) | Roche cobas® HSV 1 and 2 Test New Device (K150617) | | Intended use | The BD ProbeTec™ Herpes Simplex Viruses (HSV 1 & 2) Qx Amplified DNA Assays (HSV Qx Assays), when tested with the BD Viper™ System in Extracted Mode, use Strand | The cobas® HSV 1 and 2 Test on the cobas® 4800 system is an automated, qualitative in vitro diagnostic test, that utilizes real-time polymerase chain reaction (PCR), for the direct detection | | | Displacement Amplification technology for the direct, qualitative detection and differentiation of Herpes Simplex virus type 1 (HSV1) and Herpes Simplex virus type 2 (HSV2) DNA in clinician-collected external anogenital lesion specimens. The assays are indicated for use with symptomatic individuals to aid in the diagnosis of anogenital HSV1 and HSV2 infections. Warning: The BD ProbeTec™ Herpes Simplex Viruses (HSV 1 & 2) Qx Amplified DNA Assays (HSV Qx Assays) are not FDA cleared for use with cerebrospinal fluid (CSF). The assays are not intended to be used for prenatal screening or for individuals under the age of 17 years. | Amplification and differentiation of Herpes simplex virus 1 and 2 (HSV-1 and HSV-2) DNA in clinician-collected anogenital lesion specimens from symptomatic male and female patients. The cobas® HSV 1 and 2 Test is intended for use as an aid in diagnosis of anogenital HSV-1 and HSV-2 infections in symptomatic patients. Warning: The cobas® HSV 1 and 2 Test is not FDA cleared for use with cerebrospinal fluid (CSF) and is not intended to be used for prenatal screening or for individuals under the age of 18 years. | | Sample Types | External anogenital lesions | Same | | Assay Results | Qualitative detection and differentiation of HSV-1 and HSV-2 DNA | Same | | Detection Chemistry | Paired reporter and quencher fluorescence labeled probes using fluorescence resonance energy transfer (FRET) | Same | | Differences | | | | Characteristic | BD ProbeTec™ Herpes Simplex Viruses (HSV 1 & 2) Qx Amplified DNA Assays on the BD Viper System in Extracted Mode, (K103798) | Roche cobas® HSV 1 and 2 Test New Device (K150617) | | Amplification Technology | Strand Displacement Amplification | Real-time PCR | | Sample Preparation Procedure | Automated on BD™™ Viper™™ System in Extracted Mode | Automated on cobas® 4800 System | Similarities and Differences Between the cobas® HSV 1 and 2 Test and the Table 1: Predicate Device {8}------------------------------------------------ {9}------------------------------------------------ #### NON-CLINICAL PERFORMANCE EVALUATION 4. #### Analytical sensitivity (Limit of Detection) The analytical sensitivity (Limit of Detection or LOD) for the cobas® HSV 1 and 2 Test was determined by analyzing quantified HSV-1 and HSV-2 viral cultures diluted at multiple concentration levels into a simulated anogenital lesion swab matrix. The simulated matrix composed of mucin and human cells and mimics the effect of the clinical anogenital background for the cobas® HSV 1 and 2 Test. All levels were tested with at least 21 replicates using the full cobas® HSV 1 and 2 Test workflow across five lots of cobas® HSV 1 and 2 Test reagents. LOD for this test is defined as the target concentration which can be detected as positive in ≥ 95% of the replicates tested, based on results generated by the worst performing lot. HSV-1 Maclntyre and HSV-2 G strains were tested in the analytical sensitivity study. The cobas® HSV 1 and 2 Test LOD on these isolates is shown in Table 2. | Organism | Strain | ATCC ID | LOD (TCID50 /mL) | |----------|-----------|---------|------------------| | HSV-1 | MacIntyre | VR-539 | 0.479 | | HSV-2 | G | VR-734 | 0.112 | Table 2: cobas® HSV 1 and 2 Test LOD (Limit of Detection) ### Analytical Inclusivity Four HSV-1 strains (VR-260. VR-733. VR-735 and VR-1493) and three HSV-2 strains (VR-1779, VR-1781 and VR-540) were tested for reactivity with the cobas® HSV 1 and 2 Test. These strains were obtained from ATCC and were cultured and quantified by Virapur, LLC (California, US). Each strain was diluted in a similar fashion as in the Limit of Detection study and was tested in 40 replicates near the LoD. All strains were detected by the assay. demonstrating that the cobas 9 HSV 1 and 2 Test can detect a broad range of both HSV-1 and HSV-2 isolates. {10}------------------------------------------------ ### Precision An in-house precision study was conducted with HSV-1 and HSV-2 viruses diluted in a simulated anogenital swab matrix to concentration levels below Limit of Detection (LOD), near LOD and above LOD of the cobas® HSV 1 and 2 Test. A negative level composed of only the simulated anogenital swab matrix was also tested. The study used three unique lots of cobas® HSV 1 and 2 Test reagents and three instruments for a total of 36 runs over 12 days. A description of the precision panels and the study results are shown in Table 3. An analysis of the variance of the Ct values from valid tests was performed on positive panel members at above LOD concentrations. The analysis yielded overall CV (%) of 2.2% for HSV-1 Ct and 1.9% for HSV-2 Ct (see Table 4 and Table 5). | Panel Member | Concentration | | HSV-1 (N=72) | | | HSV-2 (N=72) | | | |-----------------|---------------|--------------|---------------------|-------------|--------------------|---------------------|-------------|--------------------| | | HSV-1 | HSV-2 | Positive Results | Hit rate | 95% 2- Sided CI | Positive Results | Hit rate | 95% 2- Sided CI | | P1 | Negative | Negative | 0 | 0% | 0 - 5% | 0 | 0% | 0 - 5% | | P2 | < LOD | < LOD | 36 | 50% | 38 - 62% | 40 | 56% | 43 - 67% | | P3 | ~ LOD | Negative | 72 | 100% | 95 - 100% | 0 | 0% | 0 - 5% | | P4 | Negative | ~ LOD | 0 | 0% | 0 - 5% | 71 | 99% | 93 - 100% | | P5 | ~3 x LOD | ~ LOD | 72 | 100% | 95 - 100% | 72 | 100% | 95 - 100% | | P6 | ~ LOD | ~ 3 x LOD | 72 | 100% | 95 - 100% | 72 | 100% | 95 - 100% | In-house precision study hit rate analysis Table 3: Table 4: Variance components analysis for precision panel at 3 x LOD (Limit of Detection) | Target | HSV Level | Mean Ct | Variance Components/Percent Contribution to Total | | | | | | |--------|--------------|---------|---------------------------------------------------|----------|------------|---------|--------|-------| | | | | Lot | Kit Size | Instrument | Run/Day | Random | Total | | HSV-1 | ~ 3 x LOD | 37.4 | 0 | 0.06 | 0 | 0.355 | 0.289 | 0.704 | | | | | 0% | 8.60% | 0% | 50.40% | 41.10% | 100% | | HSV-2 | ~ 3 x LOD | 38.2 | 0.035 | 0 | 0.049 | 0.102 | 0.345 | 0.53 | | | | | 6.50% | 0% | 9.10% | 19.30% | 65.00% | 100% | {11}------------------------------------------------ | Target | N | Mean Ct | Standard Deviation Components/CV Percent | | | | | | |--------|----|------------|------------------------------------------|----------|------------|---------|--------|-------| | | | | Lot | Kit Size | Instrument | Run/Day | Random | Total | | HSV-1 | 72 | 37.4 | 0 | 0.245 | 0 | 0.595 | 0.538 | 0.839 | | | | | 0% | 0.70% | 0% | 1.60% | 1.40% | 2.20% | | HSV-2 | 72 | 38.2 | 0.186 | 0 | 0.22 | 0.32 | 0.587 | 0.728 | | | | | 0.50% | 0% | 0.60% | 0.80% | 1.50% | 1.90% | Table 5: Standard deviations and coefficients of variation (%) analysis for precision panel at 3 x LOD (Limit of Detection) #### Competitive inhibition Panels were constructed with HSV-1 at ~ 3 x LOD (Limit of Detection), and competing HSV-2 at ~ 300 x LOD of the cobas® HSV 1 and 2 Test; and vice versa. One hundred fold higher concentration of HSV-1 did not affect the detection of HSV2 at ~ 3 x LOD concentration and one hundred fold higher concentration of HSV-2 did not affect the detection of HSV1 at ~ 3 x LOD concentration. ### Analytical specificity/Cross-reactivity and Microbial Panel The analytical specificity of the cobas® HSV 1 and 2 Test was assessed by testing a panel of organisms that could be present in anogenital swab specimens. The panel consists of 1) 71 bacteria, fungi and viruses that may be found in anogenital swab specimens, 2) human cells , and 3) high titer HSV-1 or HSV-2. Testing was performed with the organisms and human cells alone to determine the analytical specificity of the cobas® HSV 1 and 2 Test or in the presence of HSV-1 and HSV-2 at ~ 3 x LOD to assess the potential interference of the organisms and the human cells on detection of HSV-1 and HSV-2 by the cobas® HSV 1 and 2 Test. All organisms, human cells, HSV-1 and HSV-2 viruses were spiked to 1 x 106 Units*/mL or higher except for Treponema pallidum; Chlamydia trachomatis serovar H, and Mycoplasma genitalium which were spiked to lower concentrations due to stock concentrations. *All bacteria were quantified as Colony Forming Units (CFU) except Chlamydia trachomatis serovar H and Chlamydia trachomatis serovar I which were quantified as Inclusion Forming Units (IFU); Toxoplasma gondii and Treponema pallidum which were quantified as DNA copies {12}------------------------------------------------ and Trichomonas vaginalis which was quantified in cells. Cytomegalovirus (HHV5), Human Herpes Virus 6B Strain Z29, Human Herpes Virus 7 Strain SB, Echovirus 11, Human enterovirus 71 and Rubella Virus were quantified as TCID50 units; HHV-6A strain GS, HSV-1 and HSV-2 were quantified in viral particles, HIV-1 Strain IIIB and HBV were quantified in International Units (IU). HIV-2 Strain NIH-Z, Epstein-Barr Virus (HHV4), Varicella-Zoster Virus (HHV3) and HPV plasmids (HPV11, HPV18, HPV18, HPV6) were quantified as DNA copies. Two sources of Human Herpes Virus 8 were used, one was quantified as DNA copies, and the other was quantified in cells and estimated as 150 DNA copies per cell. Human Peripheral Blood Mononuclear Cells (PBMC) were quantified as number of cells. Results indicated that none of these organisms or high concentration of human cells produced false positive results when there was no HSV-1 and HSV-2 target present. None of these organisms or high concentration of human cells interfered with the detection of HSV-1 and HSV-2 targets. A high concentration of HSV-1 (1x106 vp/mL) did not produce false HSV-2 positive results and a high concentration of HSV-2 (1x106 vp/mL) did not produce false HSV-1 positive results. {13}------------------------------------------------ | Human Adenovirus type 7 | Staphylococcus aureus (MRSA) | Moraxella catarrhalis | |-------------------------------------|-----------------------------------------|------------------------------| | Cytomegalovirus (HHV5) | Staphylococcus aureus (MSSA) | Moraxella lacunata | | Epstein-Barr Virus (HHV4) | Staphylococcus epidermidis | Mycobacterium tuberculosis | | Varicella-Zoster Virus (HHV3) | Propionibacterium acnes | Mycoplasma genitalium** | | Human Herpes Virus 6A strain GS | Escherichia coli | Mycoplasma hominis | | Human Herpes Virus 6B Strain Z29 | Chlamydia trachomatis serovar H** | Neisseria gonorrhoeae | | Human Herpes Virus 7 Strain SB | Chlamydia trachomatis serovar I | Neisseria meningitidis | | Human Herpes Virus 8* | Clostridium perfringens | Prevotella melaninogenica | | Echovirus 11 | Clostridium difficile | Proteus vulgaris | | Enterovirus 71 | Corynebacterium genitalium | Pseudomonas aeruginosa | | HBV | Cryptococcus neoformans | Staphylococcus saprophyticus | | HIV-1 Strain IIIB | Enterobacter cloacae | Streptococcus agalactiae | | HIV-2 Strain NIH-Z | Enterococcus faecalis vanB | Streptococcus mitis | | HPV11 | Enterococcus faecium vanA | Streptococcus mutans | | HPV16 | Fusobacterium nucleatum | Streptococcus pneumoniae | | HPV18 | Gardnerella vaginalis | Streptococcus pyogenes | | HPV6 | Gemella haemolysans | Streptococcus salivarius | | Rubella Virus | Haemophilus ducreyi | Toxoplasma gondii | | Acinetobacter calcoaceticus | Haemophilus influenzae | Treponema pallidum** | | Acinetobacter lwoffii | Kingella kingae | Trichomonas vaginalis | | Actinomyces israelii | Klebsiella pneumoniae subsp. ozaenae | Veillonella parvula | | Alcaligenes faecalis | Lactobacillus acidophilus | PBMC (human genomic DNA) | | Bacteroides fragilis | Listeria monocytogenes | Herpes Simplex Virus-1 | | Candida albicans | Mobiluncus curtisii spp. curtisii | Herpes Simplex Virus-2 | | Candida glabrata | Mobiluncus mulieris | | #### Table 6: Cross Reactivity Panel * Two sources of Human Herpes Virus 8 were tested at 1.0 x 10 copies/mL (HHV8 viral DNA and HHV8 containing human cell line BCP-1) ** Chlamydia trachomatis serovar H was tested at 1.9 x 10° IFU/mL; Mycoplasma genitalium was tested at 1.0 x 105 CFU/mL; Treponema pallidum was tested at 9.0 x 104 copies/mL #### Interference Twenty commonly used over the counter (OTC) products and anti-viral medications, as well as whole blood, human serum albumin, urine, feces, and mucin, were tested for potential interference effects with the cobas® HSV 1 and 2 Test. All OTC products were tested at or above (20 mg or 40 mg per swab respectively for solids and100% of swab capacity for liquids) {14}------------------------------------------------ the levels that could be reasonably expected to be collected by a swab in an anogenital lesion specimen. Anti-viral medicine was tested at 3 x Cmax in the collected specimen. HSV-1 and HSV-2 were spiked to ~ 3 x LOD (Limit of Detection) of the cobas® HSV 1 and 2 Test and used as targets in the tests. No interference was observed for the OTC products except for Vagisil Crème (interference observed at 10 mg and above). For whole blood, no interference was observed up to 40% of the swab capacity; for mucin, no interference was observed up to 4.8 mg per swab; for urine, no interference was observed up to 100% of the swab capacity; for feces, no interference was observed up to 1.6 mg per swab and for human serum albumin, no interference was observed up to 16 mg per swab. These results are summarized in Table 7. | Substance/Product Name | Composition | Testing Level/Swab | |-----------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------| | Whole blood | Human whole blood | 40%, 50% | | Mucin | Mucin Type II from porcine stomach | 4.8 mg, 8 mg, 12 mg, 20 mg | | Urine | Human urine | 70%, 100% | | Feces | Human feces | 1.6 mg, 4 mg | | Human Serum Albumin | Human serum albumin, fatty acid and globulin free | 8 mg, 16 mg | | K-Y Brand Jelly (Personal Lubricant) | Glycerin, Hydroxyethylcellulose, Chlorhexidine Gluconate, Methylparaben, Sodium Hydroxide, Water | 20 mg, 40 mg | | Gynol II (Contraceptive jelly) | 3% Nonoxynol-9, Lactic Acid, Methylparaben, Povidone, Propylene Glycol, Purified Water, Sodium Carboxymethylcellulose, Sorbic Acid, Sorbitol Solution | 20 mg, 40 mg | | YeastGard Suppositories | Pulsatilla, Candida Parapsilosis, Candida Albicans, Bacillus Coagulans, Polyethylene Glycols | 20 mg, 40 mg | | Monistat 1 | 2% Miconazole nitrate, Benzoic Acid, Cetyl Alcohol, Isopropyl Myristate, Polysorbate 60, Potassium Hydroxide, Propylene Glycol, Purified Water, Stearyl Alcohol | 20 mg, 40 mg | | Monistat 3 | 4% Miconazole nitrate, Benzoic Acid, Cetyl Alcohol, Isopropyl Myristate, Polysorbate 60, Potassium Hydroxide, Propylene Glycol, Purified Water, Stearyl Alcohol | 20 mg, 40 mg | | Table 7: Interfering Substances | | | |--------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------| | Substance/Product Name | Composition | Testing Level/Swab | | VagiStat 1 | 6.5% Tioconazole, Butylated Hydroxyanisole, Magnesium Aluminium Silicate, White Petrolatum | 20 mg, 40 mg | | Clotrimazole vaginal cream | 1% Clotrimazole, Benzyl Alcohol, Cetostearyl Alcohol, Cetyl Esters Wax, 2-Octyldodecanol, Polysorbate 60, Purified Water, Sodium Phosphate Monobasic, Sorbitan Monostearate | 20 mg, 40 mg | | Preparation H Hemorrhoidal cream | 14.4% Glycerin, 0.25% Phenylephrine HCl, 1% Pramoxine HCl, 15% white Petrolatum, Aloe Barbadensis Leaf Extract, Anhydrous Citric Acid, Butylated Hydroxyanisole, Carboxymethylcellulose Sodium, Cetyl Alcohol, Citric Acid Monohydrate | 20 mg, 40 mg | | Abreva cold sore treatment | 10% Docosanol, Benzyl Alcohol, Light Mineral Oil, Propylene Glycol, Purified Water, Sucrose Distearate, Sucrose Stearate | 20 mg, 40 mg | | Releev cold sore treatment | Benzalkonium Chloride, Purified Water, Viracea, Methyl Cellulose, Methyl Paraben, Potassium Sorbate, Propyl Paraben | 20 mg, 40 mg | | Acyclovir Cream | 5% Acyclovir, Polyethylene Glycol | 20 mg, 40 mg | | Vagisil Crème | 20% Benzocaine, 3% Resorcinol, Water, Mineral Oil, Cetyl Alcohol, Propylene Glycol, Glyceryl Stearate, PEG-100 Stearate, Isopropyl Palmitate, Aloe Barbadensis Leaf Juice, Tocopheryl Acetate, Retinyl Palmitate, Zea Mays Oil | 2.5 mg, 5 mg, 10 mg, 20 mg, 40 mg | | Balneol Hygienic Cleansing lotion | Water, Mineral Oil, Propylene Glycol, Glyceryl Stearate, PEG-100 Stearate, PEG-40 Stearate, Laureth 4, PEG-4 Dilaurate, Lanolin Oil, Sodium Acetate, Carbomer 934, Triethanolamine, Methylparaben | 20 mg, 40 mg | | Vagicaine Anti-Itch Cream | 20% Benzocaine, 3% Resorcinol, Aloe barbadensis Leaf Extract, Carbomer Homopolymer Type C, Cetyl Alcohol, Cholecalciferol, Corn Oil, Glyceryl Monostearate, Isopropyl Myristate, Isopropyl Palmitate, Lanolin Alcohol, Methylparaben | 20 mg, 40 mg | | VH Essentials Douche | 3% Povidone-iodine, Purified Water, USP, Octylphenoxypolyethoxyethanol | 100% | | Denavir | 1% Penciclovir, Cetomacrogol 1000BP, Cetosteryl Alcohol, Mineral Oil, Propylene Glycol, Purified Water, White Petrolatum | 20 mg, 40 mg | | Famciclovir | Famciclovir, Hydroxypropyl Cellulose, Hydroxypropyl Methylcellulose, Lactose, Magnesium Stearate, Polyethylene Glycols, Sodium Starch Glycolate, Titanium Dioxide | 0.016 mg | | Substance/Product Name | Composition | Testing Level/Swab | | Valacyclovir | Valacyclovir Hydrochloride, Carnauba Wax, Colloidal Silicon Dioxide, Crospovidone, Hypromellose, Magnesium Stearate, Microcrystalline Cellulose, Polyethylene Glycol | 0.027 mg | | Cidofovir | Cidofovir, Sodium hydroxide, Sterile Water | 0.552 mg | | Acyclovir | Acyclovir, Magnesium Stearate, Microcrystalline Cellulose, Povidone, Sodium Starch Glycolate | 0.008 mg | {15}------------------------------------------------ {16}------------------------------------------------ ### Reproducibility The reproducibility of the cobas® HSV 1 and 2 Test on the cobas® 4800 System was established in a multi-site, investigation using contrived clinical samples evaluated across lot, site/instrument, operator, day, and run. HSV test panels were prepared by spiking HSV-1 (Maclntyre strain) and/or HSV-2 (G strain) virus into contrived sample matrix in transport media at one of three concentrations (Below LOD, 1 × LOD, and 3 × LOD); HSV-1 and HSV-2 negative panel members were included as panel member controls. In all, there were 6 members per test panel with 3 replicates per panel member included in each run, not including external positive and negative assay controls. Panels were tested at 3 sites by 2 operators per site with 1 valid run per operator per day, for 6 days per lot over 2 lots for a total of 1,296 valid tests (216 tests/panel member or 108 tests/panel member/lot). Table 8 summarizes the percent agreement (two-sided 95% exact CI) for the negative panel member and HSV-1 positive panel members. {17}------------------------------------------------ | | | | HSV-1 | |----------------------------------|------------------------------------|---------------------|-----------------| | Panel Member | Number of Valid Test Results | Agreement (n/N) | 95% CIa | | Negativeb | 216 | 100.0% (216/216) | (98.3%, 100.0%) | | Below LOD (HSV-1/HSV-2) | 216 | 63.4% (137/216) | (56.6%, 69.9%) | | 1 x LOD (HSV-1)c | 216 | 100.0% (216/216) | (98.3%, 100.0%) | | 1 x LOD (HSV-2)c | 216 | 99.5% (215/216) | (97.4%, 100.0%) | | 3 x LOD (HSV-1)/1 x LOD (HSV-2) | 216 | 100.0% (216/216) | (98.3%, 100.0%) | | 1 x LOD (HSV-1)/3 x LOD (HSV-2)c | 216 | 100.0% (216/216) | (98.3%, 100.0%) | ### Table 8: Percent agreement by panel member - HSV-1 4 95% CI = 95% exact binomial confidence interval. b Negative panel members for HSV-1: percent negative agreement was 99.8% (431/432) with 95% CI (98.7%, 100.0%). ° Panel members with 1 x LOD HSV-1: percent positive agreement was 100.0% (432/432) with 95% CI (99.1%, 100.0%). Note: Results are included as agreement when a positive panel member has a valid positive result for that analyte or when the negative panel member has a valid negative result for both analytes. CI = confidence interval; LOD = limit of detection. Table 9 presents the percent agreement (negative or positive) by lot, site/instrument, operator, and day for HSV-1 test results for each panel member. {18}------------------------------------------------ | | | | Percent Agreement (n/N)* | | | | | | | | | |----------------------------|------|------|--------------------------|---|--------------------|---|------------------|---|------------------|---|------------------| | Panel Member | | Ct | | | Lot | | Site/Inst. | | Operator | | Day | | | Mean | SD | CV % | | | | | | | | | | Negative | N/A | N/A | N/A | 1 | 100.0 (108/108) | 1 | 100.0 (72/72) | 1 | 100.0 (36/36) | 1 | 100.0 (36/36) | | | | | | 2 | 100.0 (108/108) | 2 | 100.0 (72/72) | 2 | 100.0 (36/36) | 2 | 100.0 (36/36) | | | | | | | | 3 | 100.0 (72/72) | 3 | 100.0 (36/36) | 3 | 100.0 (36/36) | | | | | | | | | | 4 | 100.0 (36/36) | 4 | 100.0 (36/36) | | | | | | | | | | 5 | 100.0 (36/36) | 5 | 100.0 (36/36) | | | | | | | | | | 6 | 100.0 (36/36) | 6 | 100.0 (36/36) | | | | | | | | | | | | | | | Below LOD (HSV-1/HSV-2) | 41.1 | 1.41 | 3.4 | 1 | 60.2 (65/108) | 1 | 56.9 (41/72) | 1 | 58.3 (21/36) | 1 | 58.3 (21/36) | | | | | | 2 | 66.7 (72/108) | 2 | 68.1 (49/72) | 2 | 55.6 (20/36) | 2 | 58.3 (21/36) | | | | | | | | 3 | 65.3 (47/72) | 3 | 63.9 (23/36) | 3 | 66.7 (24/36) | | | | | | | | | | 4 | 72.2 (26/36) | 4 | 72.2 (26/36) | | | | | | | | | | 5 | 63.9 (23/36) | 5 | 66.7 (24/36) | | | | | | | | | | 6 | 66.7 (24/36) | 6 | 58.3 (21/36) | | | | | | | | | | | | | | | 1 x LOD (HSV-1) | 38.8 | 1.18 | 3.0 | 1 | 100.0 (108/108) | 1 | 100.0 (72/72) | 1 | 100.0 (36/36) | 1 | 100.0 (36/36) | | | | | | 2 | 100.0 (108/108) | 2 | 100.0 (72/72) | 2 | 100.0 (36/36) | 2 | 100.0 (36/36) | | | | | | | | 3 | 100.0 (72/72) | 3 | 100.0 (36/36) | 3 | 100.0 (36/36) | | | | | | | | | | 4 | 100.0 (36/36) | 4 | 100.0 (36/36) | | | | | | | | | | 5 | 100.0 (36/36) | 5 | 100.0 (36/36) | | | | | | | | | | 6 | 100.0 (36/36) | 6 | 100.0 (36/36) | | | | | | | | | | | | | | #### Percent agreement by panel member for lot, site/instrument, operator, Table 9: and day - HSV-1 {19}------------------------------------------------ | | | | | Percent Agreement (n/N)* | | | | | |----------------------------------|------|------|------|--------------------------|-----------------|-----------------|-----------------|-----------------| | Panel Member | | Ct | | Lot | Site/Inst. | Operator | Day | | | | Mean | SD | CV % | | | | | | | 1 x LOD (HSV-2) | N/A | N/A | N/A | 1 | 99.1 (107/108) | 1 98.6 (71/72) | 1 97.2 (35/36) | 1 97.2 (35/36) | | | | | | 2 | 100.0 (108/108) | 2 100.0 (72/72) | 2 100.0 (36/36) | 2 100.0 (36/36) | | | | | | | 3 100.0 (72/72) | 3 100.0 (36/36) | 3 100.0 (36/36) | | | | | | | | | 4 100.0 (36/36) | 4 100.0 (36/36) | | | | | | | | | 5 100.0 (36/36) | 5 100.0 (36/36) | | | | | | | | | 6 100.0 (36/36) | 6 100.0 (36/36) | | | | | | | | | | | | | 3 x LOD (HSV-1)/ 1 x LOD (HSV-2) | 37.0 | 1.10 | 3.0 | 1 | 100.0 (108/108) | 1 100.0 (72/72) | 1 100.0 (36/36) | 1 100.0 (36/36) | | | | | | 2 | 100.0 (108/108) | 2 100.0 (72/72) | 2 100.0 (36/36) | 2 100.0 (36/36) | | | | | | | 3 100.0 (72/72) | 3 100.0 (36/36) | 3 100.0 (36/36) | | | | | | | | | 4 100.0 (36/36) | 4 100.0 (36/36) | | | | | | | | | 5 100.0 (36/36) | 5 100.0 (36/36) | | | | | | | | | 6 100.0 (36/36) | 6 100.0 (36/36) | | | | | | | | | | | | | 1 x LOD (HSV-1)/ 3 x LOD (HSV-2) | 38.7 | 1.15 | 3.0 | 1 | 100.0 (108/108) | 1 100.0 (72/72) | 1 100.0 (36/36) | 1 100.0 (36/36) | | | | | | 2 | 100.0 (108/108) | 2 100.0 (72/72) | 2 100.0 (36/36) | 2 100.0 (36/36) | | | | | | | 3 100.0 (72/72) | 3 100.0 (36/36) | 3 100.0 (36/36) | | | | | | | | | 4 100.0 (36/36) | 4 100.0 (36/36) | | | | | | | | | 5 100.0 (36/36) | 5 100.0 (36/36) | | | | | | | | | 6 100.0 (36/36) | 6 100.0 (36/36) | | * For the negative panel member, percent agreement = (number of negative results/total valid results) x 100; for the positive panel members, percent agreement = (number of positive results/total valid results) x 100. Ct = cycle threshold; CV = coefficient of variation; Inst. = instr LOD = limit of detection; N/A = not applicable; SD = standard deviation. {20}------------------------------------------------ Table 10 presents the SD and CV (%) of Ct values for HSV-1 positive panel members overall and attributable to lot, site/instrument, operator, day, and within-run. Across HSV-1 positive panel members, the total SD ranged from 1.10 to 1.41, and the total CV (%) ranged from 3.0% to 3.4%. | | | Standard Deviation and Percent Coefficients of Variation | | | | | | | | | | | | | |-------------------------------------|-----|----------------------------------------------------------|------|------|------|----------|------|------|------|------------|------|-------|------|------| | | | Site/Inst. | | Lot | | Operator | | Day | | Within-Run | | Total | | | | Panel Member | N | Mean Ct | SD | CV % | SD | CV % | SD | CV % | SD | CV % | SD | CV % | SD | CV % | | Below LOD (HSV-1/HSV-2) | 137 | 41.1 | 0.00 | 0.0 | 0.51 | 1.3 | 0.00 | 0.0 | 0.71 | 1.7 | 1.10 | 2.7 | 1.41 | 3.4 | | 1 x LOD (HSV-1) | 216 | 38.8 | 0.14 | 0.4 | 0.53 | 1.4 | 0.00 | 0.0 | 0.00 | 0.0 | 1.05 | 2.7 | 1.18 | 3.0 | | 3 x LOD (HSV-1)/ 1 x LOD (HSV-2) | 216 | 37.0 | 0.00 | 0.0 | 0.64 | 1.7 | 0.00 | 0.0 | 0.14 | 0.4 | 0.89 | 2.4 | 1.10 | 3.0 | | 1 x LOD (HSV-1)/ 3 x LOD (HSV-2) | 216 | 38.7 | 0.00 | 0.0 | 0.47 | 1.2 | 0.15 | 0.4 | 0.16 | 0.4 | 1.03 | 2.7 | 1.15 | 3.0 | Table 10: Overall mean, standard deviations, and coefficients of variation (%) for Ct values from valid results for positive panel members - HSV-1 Ct = cycle threshold; CV = coefficient of variation; Inst. = instrument; LOD = limit of detection; SD = standard deviation. In summary, the positive percent agreement for the HSV-1 positive panel member "Below LOD (HSV-1/HSV-2)" was 63.4% (95% CI: 56.6% to 69.9%) and the positive percent agreement for all other positive panel members was 100.0% (95% CI: 98.3% to 100.0%). For the negative panel members, negative percent agreement was 99.8% (95% Cl: 98.7% to 100.0%). The total SD and total %CV across all panel members were ≤ 1.41 and ≤ 3.4%, respectively. ### HSV-2 reproducibility results Table 11 summarizes the percent agreement (two-sided 95% exact CI) for the negative panel member and HSV-2 positive panel members. {21}------------------------------------------------ | | | | HSV-2 | |----------------------------------|------------------------------------|---------------------|-----------------| | Panel Member | Number of Valid Test Results | Agreement (n/N) | 95% CIa | | Negativeb | 216 | 100.0% (216/216) | (98.3%, 100.0%) | | Below LOD (HSV-1/HSV-2) | 216 | 56.5% (122/216) | (49.6%, 63.2%) | | 1 x LOD (HSV-1)b | 216 | 100.0% (216/216) | (98.3%, 100.0%) | | 1 x LOD (HSV-2)c | 216 | 100.0% (216/216) | (98.3%, 100.0%) | | 3 x LOD (HSV-1)/1 x LOD (HSV-2)c | 216 | 100.0% (216/216) | (98.3%, 100.0%) | | 1 x LOD (HSV-1)/3 x LOD (HSV-2) | 216 | 100.0% (216/216) | (98.3%, 100.0%) | ### Table 11: Percent agreement by panel member - HSV-2 4 95% CI = 95% exact binomial confidence interval. b Negative panel members for HSV-2: percent negative agreement was 100.0% (432/432) with 95% CI (99.1%, 100.0%). & Panel members with 1 x LOD HSV-2: percent positive agreement was 100.0% (432/432) with 95% CI of (99.1%, 100.0%). Note: Results are included as agreement when a positive panel member has a valid result of positive for that analyte or when the negative panel member has a valid result of negative for both analytes. CI = confidence interval; LOD = limit of detection. {22}------------------------------------------------ Table 12 presents the percent agreement (negative and positive) by lot, site/instrument, operator, and day for HSV-2 test results for each panel member. | Table 12: Percent agreement by panel member for lot, site/instrument, operator, and day | |-----------------------------------------------------------------------------------------| | HSV-2 | | | | | | Percent Agreement (n/N)* | | | | | | |-------------------------------------|------|------|------|--------------------------|--------------------------|-----------------------|-----------------------|-----------------------|---| | Panel Member | Ct | | | Lot | Site/Inst. | Operator | Day | | | | | Mean | SD | CV% | | | | | | | | Negative | N/A | N/A | N/A | 1 | 100.0 (108/108) | 100.0 (36/36) | 100.0 (36/36) | 1 | 1 | | | | | | 2 | 100.0 (108/108) | 100.0 (36/36) | 100.0 (36/36) | 2 | 2 | | | | | | | | 100.0 (36/36) | 100.0 (36/36) | 3 | 3 | | | | | | | | 100.0 (36/36) | 100.0 (36/36) | 4 | 4 | | | | | | | | 100.0 (36/36) | 100.0 (36/36) | 5 | 5 | | | | | | | | 100.0 (36/36) | 100.0 (36/36) | 6 | 6 | | Below LOD (HSV-1/HSV-2) | 40.3 | 0.89 | 2.2 | 1 | 51.9 (56/108) | 58.3 (21/36) | 55.6 (20/36) | 1 | 1 | | | | | | 2 | 61.1 (66/108) | 72.2 (26/36) | 50.0 (18/36) | 2 | 2 | | | | | | | | 58.3 (21/36) | 47.2 (17/36) | 3 | 3 | | | | | | | | 38.9 (14/36) | 66.7 (24/36) | 4 | 4 | | | | | | | | 61.1 (22/36) | 63.9 (23/36) | 5 | 5 | | | | | | | | 50.0 (18/36) | 55.6 (20/36) | 6 | 6 | | 1 x LOD (HSV-1) | N/A | N/A | N/A | 1 | 100.0 (108/108) | 100.0 (36/36) | 100.0 (36/36) | 1 | 1 | | Panel Member | Ct | | | Percent Agreement (n/N)* | | | | | | | | Mean | SD | CV % | Lot | Site/Inst. | Operator | Day | | | | | | | | 2 (108/108) | 2 100.0 (72/72) | 3 100.0 (36/36) | 2 100.0 (36/36) | | | | | | | | | 3 100.0 (72/72) | 4 100.0 (36/36) | 3 100.0 (36/36) | | | | | | | | | | 5 100.0 (36/36) | 4 100.0 (36/36) | | | | | | | | | | 6 100.0 (36/36) | 5 100.0 (36/36) | | | | 1 x LOD (HSV-2) | 39.0 | 0.92 | 2.3 | 1 100.0 (108/108) | 1 100.0 (72/72) | 1 100.0 (36/36) | 1 100.0 (36/36) | | | | | | | | 2 100.0 (108/108) | 2 100.0 (72/72) | 2 100.0 (36/36) | 2 100.0 (36/36) | | | | | | | | | 3 100.0 (72/72) | 3 100.0 (36/36) | 3 100.0 (36/36) | | | | | | | | | | 4 100.0 (36/36) | 4 100.0 (36/36) | | | | | | | | | | 5 100.0 (36/36) | 5 100.0 (36/36) | | | | | | | | | | 6 100.0 (36/36) | 6 100.0 (36/36) | | | | 3 x LOD (HSV-1)/ 1 x LOD (HSV-2) | 38.8 | 0.86 | 2.2 | 1 100.0 (108/108) | 1 100.0 (72/72) | 1 100.0 (36/36) | 1 100.0 (36/36) | | | | | | | | 2 100.0 (108/108) | 2 100.0 (72/72) | 2 100.0 (36/36) | 2 100.0 (36/36) | | | | | | | | | 3 100.0 (72/72) | 3 100.0 (36/36) | 3 100.0 (36/36) | | | | | | | | | | 4 100.0 (36/36) | 4 100.0 (36/36) | | | | | | | | | | 5 100.0 (36/36) | 5 100.0 (36/36) | | | | | | | | | | 6 100.0 (36/36) | 6 100.0 (36/36) | | | | | | Ct | | | Percent Agreement (n/N)* | | | | | | Panel Member | | Mean | SD | CV % | Lot | Site/Inst. | Operator | Day | | | 1 x LOD (HSV-1)/ 3 x LOD (HSV-2) | | 37.8 | 0.73 | 1.9 | 1 100.0 (108/108) | 1 100.0 (72/72) | 1 100.0 (36/36) | 1 100.0 (36/36) | | | | | | | | 2 100.0 (108/108) | 2 100.0 (72/72) | 2 100.0 (36/36) | 2 100.0 (36/36) | | | | | | | | | 3 100.0 (72/72) | 3 100.0 (36/36) | 3 100.0 (36/36) | | | | | | | | | | 4 100.0 (36/36) | 4 100.0 (36/36) | | | | | | | | | | 5 100.0 (36/36) | 5 100.0 (36/36) | | | | | | | | | | 6 100.0 (36/36) | 6 100.0 (36/36) | | {23}------------------------------------------------ {24}------------------------------------------------ * For the negative panel member, percent agreement = (number of negative results/total valid results) x 100; for the positive panel members, percent agreement = (number of positive results/total valid results) x 100. Ct = cycle threshold; CV = coefficient of variation; Inst = instrument; LOD = limit of detection; N/A = not applicable; SD = standard deviation. {25}------------------------------------------------ Table 13 presents the SD and CV (%) of Ct values for HSV-2 positive panel members overall and attributable to lot, site/instrument, operator, day, and within-run. Across HSV-2 positive panel members, the total SD ranged from 0.73 to 0.92, and the total CV (%) ranged from 1.9% to 2.3%. | Table 13 Overall mean, standard deviations, and coefficients of variation (%) for Ct values | |---------------------------------------------------------------------------------------------| | from valid results for positive panel members - HSV-2 | | | | | Standard Deviation and Percent Coefficient of Variation | | | | | | | | | | | | |-------------------------------------|-----|--…