(242 days)
No
The device description and performance studies focus on standard PCR technology and data analysis methods, with no mention of AI or ML.
No
This device is an in vitro diagnostic test for detecting HSV-1 and HSV-2 DNA, meaning it aids in diagnosis rather than providing treatment or therapy.
Yes
The "Intended Use / Indications for Use" section explicitly states that the device is "a real-time polymerase chain reaction (PCR) based qualitative in vitro diagnostic test for the direct detection and differentiation of Herpes Simplex Virus 1 and 2 (HSV-2) DNA... This test is an aid in the differential diagnosis of HSV-1 and HSV-2 infections." This directly indicates its function as a diagnostic tool.
No
The device description clearly outlines a system comprised of three major processes involving automated sample preparation, PCR amplification, and real-time detection using fluorescent probes. These processes inherently involve hardware components and reagents, not just software.
Yes, this device is an IVD (In Vitro Diagnostic).
The Intended Use statement explicitly states that the HSV 1&2 ELITe MGB® Assay is a "qualitative in vitro diagnostic test".
The Device Description further reinforces this by describing it as a "qualitative in vitro diagnostic Real-Time PCR Assay".
The entire description details a test performed on biological samples (swab specimens) outside of the body to detect and differentiate specific analytes (HSV-1 and HSV-2 DNA) for diagnostic purposes. This aligns perfectly with the definition of an In Vitro Diagnostic device.
N/A
Intended Use / Indications for Use
The HSV 1&2 ELITe MGB® Assay is a real-time polymerase chain reaction (PCR) based qualitative in vitro diagnostic test for the direct detection and differentiation of Herpes Simplex Virus 1 and 2 (HSV-2) DNA in cutaneous or mucocutaneous lesion swab specimens from patients with signs and symptoms of HSV-2 infection. This test is an aid in the differential diagnosis of HSV-1 and HSV-2 infections.
The HSV 1&2 ELITe MGB Assay is not FDA cleared for use with cerebrospinal fluid (CSF) specimens. The assay is not intended to be used for prenatal screening or for screening blood or blood products.
Product codes
PGI, OOI
Device Description
The HSV 1&2 ELITe MGB Assay is a qualitative in vitro diagnostic Real-Time PCR Assay for the direct detection of Herpes Simplex Virus (HSV) DNA (glycoprotein D gene for HSV-1 and glycoprotein G gene for HSV-2) in symptomatic male and female patients using DNA purified from swab specimens collected from individuals with cutaneous or mucocutaneous herpetic lesions.
The HSV 1&2 ELITe MGB Assay system is comprised of three major processes: (1) automated preparation of unprocessed sample to extract nucleic acids from primary swab specimens using the ELITe InGenius SP 200 Extraction Cartridge, (2) PCR amplification of target DNA sequences using HSV-1 and HSV-2 specific primers, and (3) real-time detection of 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 extraction and monitors the integrity of the reagents, equipment function, and the presence of inhibitors in the samples. A positive signal in the Internal Control channel in the absence of HSV DNA indicates that the PCR has not been inhibited.
The amplification reagents, Positive Control and Internal Control are packaged as part of the HSV 1&2 ELITe MGB Assay.
Mentions image processing
Not Found
Mentions AI, DNN, or ML
Not Found
Input Imaging Modality
Not Found
Anatomical Site
cutaneous or mucocutaneous lesion swab specimens
Indicated Patient Age Range
Not Found
Intended User / Care Setting
Not Found
Description of the training set, sample size, data source, and annotation protocol
Not Found
Description of the test set, sample size, data source, and annotation protocol
A total of 1,174 left-over prospectively collected archived swab samples from cutaneous (546) and mucocutaneous (628) lesions from symptomatic patients were collected and evaluated in the study.
The samples were tested with HSV 1&2 ELITe MGB Assay and the Composite Reference Method. Out of the 1,174 tested samples were found invalid by the ELITe MGB Assay and were excluded from the performance analysis tables.
Out of the 1172 remaining samples 1 additional invalid sample result for HSV1 and 2 additional invalid sample results for HSV2 by the composite reference method were removed from the performance analysis tables.
Therefore for HSV1, 1171 samples analyzed and for HSV2 1170 samples were analyzed.
The composite reference method for the clinical performance evaluation consisted of an FDA cleared assay and a validated HSV 1&2 PCR followed by bi-directional sequencing of gel electrophoresis-positive samples. A positive result by the composite reference method is defined as a positive by the FDA cleared PCR or the validated sequencing. Two negative results are needed to confirm a negative.
Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)
Analytical Performance (Limit of Detection, Analytical Reactivity (Inclusivity), Analytical Specificity (Cross-Reactivity), Microbial Interference, Competitive Interference of HSV-1 and HSV-2, Interfering Substances, Carry-Over Contamination, Sample Stability, Reagent Stability):
- Limit of Detection (LoD): Determined using Probit (Logit) Data Analysis software. LoD for HSV-1 MacIntyre strain was 59 TCID50/mL and for HSV-2 MS strain was 5.4 TCID50/mL. Verified by testing at least 20 replicates for each strain.
- Analytical Reactivity (Inclusivity): Tested on 44 well-characterized HSV-1 and HSV-2 isolates. All HSV-1 and HSV-2 tested isolates were detected at concentrations of 16.2 - 354 TCID50/mL.
- Analytical Specificity (Cross-Reactivity): NCBI BLAST in silico analysis showed no sufficient sequence homology for cross-reactivity with non-HSV organisms. 49 potential cross-reactants were evaluated by testing samples with required concentrations; no cross-reactivity was observed.
- Microbial Interference: Evaluated in the presence of HSV-1 or HSV-2 spiked at 3xLoD and 49 organisms. None of the non-target organisms interfered with detection of HSV-1 or HSV-2, except for amplification of HSV-1 being completely inhibited in the presence of HSV-2 in titers of 1x10^3 or higher. This interference is noted as a limitation.
- Competitive Interference of HSV-1 and HSV-2: High concentration of HSV-2 (1x10^3, 1x10^4, 1x10^5 TCID50/mL) interfered with the detection of low-level HSV-1 (177 TCID50/mL), with detection rates of 1/3, 1/3, 0/3 respectively. No competitive interference of HSV-2 with HSV-1 was observed at any level. No competitive interference was observed when both strains were tested at similar or equal concentrations (3xLoD, 1x10^3, 1x10^5).
- Interfering Substances: 33 potential interfering substances (e.g., blood, Acyclovir, lubricants, common household products) were individually spiked into negative UTM matrix containing HSV-1 and HSV-2 isolates at 3xLoD. No interference was observed, and no invalid results were generated.
- Carry-Over Contamination: Tested by performing 5 integrated checkerboard runs with high positive HSV-1 samples and negative samples. No carry-over and cross-contamination were observed, with an overall percent agreement of 100% for positive and negative samples.
- Sample Stability:
- Refrigerated Stability: Samples in UTM, M4, M4RT, M5, and M6 media were stable for 1 week at +4°C (tested at 3xLoD, 1x10^3, and 1x10^5 TCID50/mL).
- Frozen Stability: Clinical samples stored at -80°C (630 reproducibility samples (minimum 210 per testing site). Tested at 3 sites by 2 operators on 10 nonconsecutive days using a single lot. Lot-to-Lot variability assessed at EGI MDx using 3 lots.
- Key Results:
- Highest Site-to-Site variability (based on Ct values): 2.19%.
- Highest Lot variability: 0.23%.
- Highest Operator variability: 0.93% for Moderate Positive panel members.
- All expected positivity rates for panels are either 100.0% or within the 95% Confidence Interval for reproducibility.
HSV-2 Contrived Oral Panel Study:
- Sample Size: 75 individual negative cheek swab samples in UTM spiked with HSV-2 at various concentrations (3×LoD, 8×LoD, 40×LoD, 200×LoD and 1000×LoD, 10 of each), 10 HSV-1 Positive samples spiked at 10×LoD and 15 HSV-1/HSV-2 Negative Oral Samples.
- Key Results: 49 out of 50 oral HSV-2 contrived samples were positive (98% detection). All 10 HSV-1 Positive samples confirmed 100% positivity.
Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)
HSV-1 in Cutaneous Lesion Samples:
- PPA: 98.7%
- NPA: 98.5%
HSV-1 in Mucocutaneous Lesion Samples:
- PPA: 99.2%
- NPA: 97.6%
HSV-2 in Cutaneous Lesion Samples:
- PPA: 96.2%
- NPA: 98.6%
HSV-2 in Mucocutaneous Lesion Samples:
- PPA: 97.6%
- NPA: 98.2%
Predicate Device(s)
Reference Device(s)
Not Found
Predetermined Change Control Plan (PCCP) - All Relevant Information
Not Found
§ 866.3309 Herpes virus nucleic acid-based cutaneous and mucocutaneous lesion panel.
(a)
Identification. A herpes virus nucleic acid-based cutaneous and mucocutaneous lesion panel is a qualitative in vitro diagnostic device intended for the simultaneous detection and differentiation of different herpes viruses in cutaneous and mucocutaneous lesion samples from symptomatic patients suspected of Herpetic infections. Negative results do not preclude infection and should not be used as the sole basis for treatment or other patient management decisions. The assay is not intended for use in cerebrospinal fluid samples.(b)
Classification. Class II (special controls). The special controls for this device are:(1) Premarket notification submissions must include detailed documentation for the device description, including the device components, ancillary reagents required but not provided, and a detailed explanation of the methodology including primer design and selection.
(2) Premarket notification submissions must include detailed documentation from the following analytical and clinical performance studies: Analytical sensitivity (Limit of Detection), reactivity, inclusivity, precision, reproducibility, interference, cross reactivity, carry-over, and cross contamination.
(3) Premarket notification submissions must include detailed documentation of a clinical study using lesion samples in which Herpes Simplex Virus 1, Herpes Simplex Virus 2, or Varicella Zoster Virus DNA detection was requested. The study must compare the device performance to an appropriate well established reference method.
(4) A detailed explanation of the interpretation of results and acceptance criteria must be included in the device's 21 CFR 809.10(b)(9) compliant labeling.
(5) The device labeling must include a limitation statement that reads: “The device is not intended for use with cerebrospinal fluid or to aid in the diagnosis of HSV or VZV infections of the central nervous system (CNS).”
(6) Premarket notification submissions must include quality assurance protocols and a detailed documentation for device software, including, but not limited to, standalone software applications and hardware-based devices that incorporate software.
(7) The risk management activities performed as part of the manufacturer's 21 CFR 820.30 design controls must document an appropriate end user device training program that will be offered as part of efforts to mitigate the risk of failure to correctly operate the instrument.
0
Image /page/0/Picture/0 description: The image shows the logo of the U.S. Food and Drug Administration (FDA). On the left is the Department of Health & Human Services logo. To the right of that is the FDA logo, which is a blue square with the letters "FDA" in white. To the right of the blue square is the text "U.S. FOOD & DRUG ADMINISTRATION" in blue.
October 29, 2018
ELITechGroup Walt Mahoney Regulatory Affairs Manager 21720 23rd Drive SE Suite 150 Bothell, Washington 98021
Re: K180559
Trade/Device Name: HSV 1 & 2 ELITe MGB Kit; ELITe InGenius Regulation Number: 21 CFR 866.3309 Regulation Name: Herpes virus nucleic acid-based cutaneous and mucocutaneous lesion panel Regulatory Class: Class II Product Code: PGI, OOI Dated: January 17, 2018 Received: March 1, 2018
Dear Walt Mahoney:
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. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database located at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. 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
1
requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803) for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/CombinationProducts/GuidanceRegulatoryInformation/ucm597488.html; good manufacturing practice requirements as set forth in the quality systems (OS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions 531-542 of the Act); 21 CFR 1000-1050.
Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR 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 comprehensive regulatory information about medical devices and radiation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/) and CDRH Learn (http://www.fda.gov/Training/CDRHLearn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (http://www.fda.gov/DICE) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).
Sincerely,
Steven R. Gitterman -S for
Uwe Scherf, 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) K180559
Device Name HSV 1&2 ELITe MGB Assay
Indications for Use (Describe)
HSV 1&2 ELITe MGB Assay
The HSV 1&2 ELITe MGB® Assay is a real-time polymerase chain reaction (PCR) based qualitative in vitro diagnostic test for the direct detection and differentiation of Herpes Simplex Virus 1 and 2 (HSV-2) DNA in cutaneous or mucocutaneous lesion swab specimens from patients with signs and symptoms of HSV-2 infection. This test is an aid in the differential diagnosis of HSV-1 and HSV-2 infections.
The HSV 1&2 ELITe MGB Assay is not FDA cleared for use with cerebrospinal fluid (CSF) specimens. The assay is not intended to be used for prenatal screening or for screening blood or blood products.
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
510(k) Summary
ELITe MGB HSV 1&2 Assay on the ELITe InGenius system
| 1. | Date: | September 25th, 2018 | ||
|---|---|---|---|---|
| 2. | Submitter: | ELITechGroup Inc. Molecular Diagnostics | ||
| 21720 23rd Drive SE, Suite 150 | ||||
| Bothell, WA 98021 | ||||
| 3. | Contact Person: | Walt Mahoney | ||
| 21720 23rd Dr. SE, Suite 150 | ||||
| Bothell, WA 98021 | ||||
| Phone: | 425-482-5173 | |||
| Fax: | 425-482-5550 | |||
| Email: | w.mahoney@elitechgroup.com | |||
| 4. | Device Description: | K180559 | ||
| HSV 1&2 ELITE MBG Assay | ||||
| Class II | ||||
| PGI | ||||
| 21CFR 866.3309 | ||||
| 5. | Predicate Devices: | K151906 | ||
| LUMINEX Corporation | ||||
| ARIES® HSV 1&2 Assay |
4
Intended Use 6.
HSV 1&2 ELITe MGB Assay The HSV 1&2 ELITe MGB® Assay is a real-time polymerase chain reaction (PCR) based qualitative in vitro diagnostic test for the direct detection and differentiation of Herpes Simplex Virus 1 and 2 (HSV-1 and HSV-2) DNA in cutaneous or mucocutaneous lesion swab specimens from patients with signs and symptoms of HSV-1 or HSV-2 infection. This test is an aid in the differential diagnosis of HSV-1 and HSV-2 infections.
The HSV 1&2 ELITe MGB Assay is not FDA cleared for use with cerebrospinal fluid (CSF) specimens. The assay is not intended to be used for prenatal screening or for screening blood or blood products.
Special instrument requirements: for use on the ELITe InGenius system
7. Device Descriptions
HSV 1&2 ELITe MGB Assay
The HSV 1&2 ELITe MGB Assay is a qualitative in vitro diagnostic Real-Time PCR Assay for the direct detection of Herpes Simplex Virus (HSV) DNA (glycoprotein D gene for HSV-1 and glycoprotein G gene for HSV-2) in symptomatic male and female patients using DNA purified from swab specimens collected from individuals with cutaneous or mucocutaneous herpetic lesions.
The HSV 1&2 ELITe MGB Assay system is comprised of three major processes: (1) automated preparation of unprocessed sample to extract nucleic acids from primary swab specimens using the ELITe InGenius SP 200 Extraction Cartridge, (2) PCR amplification of target DNA sequences using HSV-1 and HSV-2 specific primers, and (3) real-time detection of 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 extraction and monitors the integrity of the reagents, equipment function, and the presence of inhibitors in the samples. A positive signal in the Internal Control channel in the absence of HSV DNA indicates that the PCR has not been inhibited.
The amplification reagents, Positive Control and Internal Control are packaged as part of the HSV 1&2 ELITe MGB Assay.
8. Substantial Equivalence Information
Device Name: HSV 1&2 ELITe MGB Assay Predicate Device Name: Aries HSV 1&2 Assay, 510k number: K151906
Comparison with predicate
5
| Similarities | ||
|---|---|---|
| Item | New Device | |
| HSV 1&2 ELITE MGB Assay | Predicate Device | |
| Aries HSV 1&2 Assay, K151906 | ||
| Intended | ||
| Use/Indicat | ||
| ions for | ||
| Use | The HSV 1&2 ELITE MGB® Assay is a | |
| real-time polymerase chain reaction | ||
| (PCR) based qualitative in vitro | ||
| diagnostic test for the direct detection | ||
| and differentiation of Herpes Simplex | ||
| Virus 1 and 2 (HSV-1 and HSV-2) DNA | ||
| in cutaneous or mucocutaneous lesion | ||
| swab specimens from patients with | ||
| signs and symptoms of HSV-1 or HSV- | ||
| 2 infection. This test is an aid in the | ||
| differential diagnosis of HSV-1 and | ||
| HSV-2 infections. | ||
| The HSV 1&2 ELITE MGB Assay is not | ||
| FDA Cleared for use with cerebrospinal | ||
| fluid (CSF) specimens. The assay is not | ||
| intended to be used for prenatal | ||
| screening or for screening blood or | ||
| blood products. | The ARIES® HSV 1&2 Assay is a real- | |
| time polymerase chain reaction (PCR) | ||
| based qualitative in vitro diagnostic test | ||
| for the direct detection and | ||
| differentiation of Herpes Simplex Virus | ||
| 1 and 2 (HSV 1 and HSV 2) DNA in | ||
| cutaneous or mucocutaneous lesion | ||
| specimens from symptomatic patients. | ||
| The test is indicated for use as an aid in | ||
| diagnosis of HSV infection in | ||
| symptomatic patients. The ARIES® | ||
| HSV 1&2 Assay is indicated for use on | ||
| the ARIES® System. | ||
| WARNING: The ARIES® HSV 1&2 | ||
| Assay is not FDA cleared for use with | ||
| cerebrospinal fluid (CSF). The assay is | ||
| not intended to be used for prenatal | ||
| screening. | ||
| Specimen | ||
| types | Male and female cutaneous and | |
| mucocutaneous lesion swab specimens | Same | |
| Major | ||
| Technology | Qualitative real-time PCR | Same |
| Sample | ||
| extraction | ||
| method | Automated sample extraction | Same |
| Assay | ||
| results | Qualitative detection and differentiation | |
| of HSV-1 and HSV-2 DNA | Same |
Differences
| Differences | ||
|---|---|---|
| Parameter | New Device | |
| HSV 1&2 ELITe MGB Assay | Predicate Device | |
| Aries HSV 1&2 Assay, K151906 | ||
| Sample extraction & | ||
| Amplification | ||
| Instrumentation | ELITe InGenius system | ARIES system |
| Analyte | ||
| measures | DNA sequences from HSV-1 | |
| glycoprotein D gene and HSV-2 | ||
| glycoprotein G gene. | DNA sequences from Herpes Simplex | |
| Virus type 1 (HSV-1) and Herpes | ||
| Simplex Virus type 2 (HSV-2) | ||
| Detection | ||
| Method | Multiplex assay with paired reporter | |
| and quencher fluorescence labeled | ||
| probes and different reporter dyes | ||
| for each target. Measures increase | ||
| in assay fluorescence with each | ||
| PCR cycle. | Pairs fluorescent-labeled primers with | |
| quencher labeled nucleotides. | ||
| Measures decrease in assay | ||
| fluorescence with each PCR cycle. |
6
9. Instrumentation/Software:
The HSV 1&2 ELITe MGB Assay is used on the ELITe InGenius system. The ELITe InGenius system is a bench top instrument integrating all required hardware, reagent and software components to perform nucleic acid sample preparation and real-time PCR operations.
The ELITe InGenius system can process from 1 to 12 samples in 12 parallel tracks, and samples may be loaded in primary tubes or in secondary tubes provided. The system utilizes a universal, cassette-based process for sample extraction, and allows for multiple and independent PCRs to be performed from a single nucleic acid eluate.
The system can operate in three different modes: nucleic acid extraction only, PCR amplification only, or nucleic acid extraction with PCR amplification.
10 Performance Characteristics - Analytical Performance
a. Limit of Detection
The analytical sensitivity of the HSV 1&2 ELITe MGB Assay was determined using 4 HSV strains (two for each target). Quantitated viral strains were obtained and diluted with HSV neqative pooled human cheek matrix in Universal Transport Medium (UTM) to values spanning the range of approximately .05 to 1000 TCIDsg/mL (depending on the strain). All dilutions were tested, and the limit of detection (LoD) was determined using Probit (Logit) Data Analysis software (Analyse-it for Microsoft Excel v4.80.2, Logistic Function model). LoD for each strain represents the lowest viral titer in TCID50/mL at which a positive result will be obtained with at least 95% confidence. LoD for each strain was then verified by testing at least 20 replicates. Results indicate that, depending on the strain, the HSV 1&2 ELITe MGB Assay will produce a positive result with 95% confidence for a swab eluate containing 59 (HSV-1; HSV-1 MacIntyre Strain) and/or 5.4 (HSV-2; HSV-2 MS Strain) TCIDg6/mL.
| Organism | Isolate/Strain | Cell
Line | Qualitative
results
#detected/Total | Mean CT ±SD
from detected
replicates | 1×LoD
TCID50/mL |
|----------|------------------------------|--------------|-------------------------------------------|--------------------------------------------|--------------------|
| HSV-1 | MacIntyre
strain | Vero | 20/20 | 37.91 ± 0.69 | 59.0
TCID50/mL |
| HSV-1 | Isolate #15
(Zeptometrix) | Vero | 20/20 | 39.94 ± 0.95 | 1.5
TCID50/mL |
| HSV-2 | MS strain | Vero | 20/20 | 37.90 ± 0.92 | 5.4
TCID50/mL |
| HSV-2 | Isolate #2
(Zeptometrix) | Vero | 20/20 | 38.67 ± 1.03 | 0.3
TCID50/mL |
Limit of Detection Results
A swab elution efficiency study was also performed by using the same HSV negative pooled human cheek swab matrix and the HSV-1 MacIntyre strain used to verify the LoB. This study showed 100% elution efficiency from the Copan reqular flocked swab compared with the same volume of material directly spiked into UTM. Therefore, LoD values in TCID56/mL units will be directly proportional (with a constant = 1) to the LoD in TCIDs /swab units, depending only on the volume of the media in the collection device.
b. Assay Cut Off
7
The assay cut-off analysis was performed on a separate set of 141 clinical samples collected from 3 clinical sites. Each clinical sample was evaluated using HSV 1&2 ELITe MGB Assay in conjunction with the ELITe InGenius instrument and a composite reference method (FDA-cleared real-time PCR assay combined with PCR amplification and bidirectional sequencinq). Both targets in clinical samples were detected up to cycle 45. Therefore C- of 45 was established as a diagnostic assay cut-off for both HSV-1 and HSV-2 targets.
c. Analytical Reactivity (Inclusivity)
Performance of the HSV 1&2 ELITe MGB Assay was tested on 44 well characterized HSV-1 and HSV-2 isolates, which were obtained through commercial means. All strains were tested with the HSV 1&2 ELITE MGB Assay as spiked samples in UTM that had been prepared at 3×LOD level (177 TCIDsp/mL for HSV-1 and 16.2 TCID50/mL for HSV-2). For HSV-1 and HSV-2 viral isolates not detected (negative) at 3×LoD concentrations, 2× incremental concentrations were tested, and the lowest detectable level was determined, and the final test concentration is reported. All of the HSV-1 and HSV-2 tested isolates were detected by the HSV 1&2 ELITe MGB Assay at concentrations of 16.2 - 354 TCID50/mL
| # | Isolate | Estimated
1×LoD
(TCID50/mL) | ×LoD
Tested | Final Test
Conc.
(TCID50/mL) | Positivity |
|----|---------------------------|--------------------------------------------------------------------------------------------------------------------------------|----------------|------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| 1 | HSV-1 MacIntyre
Strain | 59 | 3× | 177 | 3/3 |
| 2 | HSV-1 Isolate #1 | ട് ക | રૂપ્ | 177 | ર્ડારે |
| 3 | HSV-1 Isolate #2 | રેત્વે છે. વિત્તર પ્રદેશના પશુપાલન છે. આ ગામના લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામમાં મુખ્યત્વે ખેત-ઉત | 3× | 177 | રૂડિયારે તે જેવી સાચના સાચના દિવેલી છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામના |
| 4 | HSV-1 Isolate #3 | ട് വ | 3× | 177 | 3/3 |
| 5 | HSV-1 Isolate #4 | ട്ടു | 3× | 177 | ર્ડારે |
| 6 | HSV-1 Isolate #5 | રેત્વે છે. | રૂપ્ર | 177 | ર્ડારે |
| 7 | HSV-1 Isolate #6 | 59 | રૂપ્ર | 177 | 0/3 |
| | | ನಿರಿ | 6× | 354 | રાજ |
| 8 | HSV-1 Isolate #7 | રેત્વે છે. વિત્તર પ્રદેશના પશુપાલન છે. આ ગામના લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામમાં મુખ્યત્વે ખેત-ઉત | 3× | 177 | ર્ડારે |
| 9 | HSV-1 Isolate #8 | 59 | 3× | 177 | ર્ડારે |
| 10 | HSV-1 Isolate #9 | 59 | રૂપ્ર | 177 | ર્ડારે |
| 11 | HSV-1 Isolate #10 | 59 | 3× | 177 | 3/3 |
| 12 | HSV-1 Isolate #11 | 59 | 3× | 177 | 3/3 |
| 13 | HSV-1 Isolate #12 | રેત્વે છે. | 3× | 177 | ર્ડારે |
| 14 | HSV-1 Isolate #13 | ട് ഒര | 3× | 177 | 3/3 |
| 15 | HSV-1 Isolate #14 | 59 | 3× | 177 | 3/3 |
| 16 | HSV-1 Isolate #15 | 59 | 3× | 177 | 3/3 |
| 17 | HSV-1 Isolate #16 | 59 | 3× | 177 | રૂડિયારે તે જેવી સાચના સાચના દિવેલી છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામના |
| 18 | HSV-1 Isolate #17 | રેત્વે છે. | 3× | 177 | ર્ડારે |
| 19 | HSV-1 Isolate #18 | 59 | 3× | 177 | રૂડિયારે તે જેવી સાચના સાચના દિવેલી છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામના |
| 20 | HSV-1 Isolate #19 | ട് ക | 3× | 177 | રૂડિયારે તે જેવી સાચના સાચના દિવેલી છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામના |
| 21 | | ട്ടു | 3× | 177 | 0/3 |
| | HSV-1 Isolate #20 | 59 | 6× | 354 | રૂડિયારે તે જેવી સાચના સાચના દિવેલી છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામના |
| 22 | HSV-1 Isolate #21 | ട് ഒര | રૂપ્ર | 177 | રાજ |
| 23 | HSV-2 MS Strain | 5.4 | 3× | 16.2 | રૂડિયારે તે જેવી સાચના સાચના દિવેલી છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામના |
| 24 | HSV-2 Isolate #1 | 5.4 | 3× | 16.2 | રૂડિયારે તે જેવી સાચના સાચના દિવેલી છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામના |
| 25 | HSV-2 Isolate #2 | 5.4 | 3× | 16.2 | ર્ડારે |
| 26 | HSV-2 Isolate #3 | 5.4 | રજ | 16.2 | 3/3 |
Summary of Inclusivity Results
8
| 27 | HSV-2 Isolate #4 | 5.4 | રૂપ્ | 16.2 | 3/3 |
|---|---|---|---|---|---|
| 28 | HSV-2 Isolate #5 | 5.4 | 3× | 16.2 | રીકે |
| 29 | HSV-2 Isolate #6 | 5.4 | 3× | 16.2 | રૂડિયારે તે જેવી સાચના સાચના દિવેલી છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામના |
| 30 | HSV-2 Isolate #7 | 5.4 | રૂપ્ | 16.2 | રૂડિયારે તે જેવી સાચના સાચના દિવેલી છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામના |
| 5.4 | 3× | 16.2 | 2/3 | ||
| 31 | |||||
| HSV-2 Isolate #8 | 5.4 | રૂપ્ | 16.2 | 3/3 | |
| 32 | HSV-2 Isolate #9 | 5.4 | 3× | 16.2 | રૂડિયારે તે જેવી સાચના સાચના દિવેલી છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામના |
| 33 | HSV-2 Isolate #10 | 5.4 | 3× | 16.2 | ર્ડારે |
| 34 | 5.4 | 3× | 16.2 | 2/3 | |
| HSV-2 Isolate #11 | 5.4 | 6× | 32.4 | રૂડિયારે તે જેવી સાચના સાચના દિવેલી છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામના | |
| 35 | 5.4 | 3× | 16.2 | 113 | |
| HSV-2 Isolate #12 | 5.4 | 6× | 32.4 | 3/3 | |
| 5.4 | રૂપ્ | 16.2 | 0/3 | ||
| 5.4 | 6× | 32.4 | 2/3 | ||
| 36 | HSV-2 Isolate #13 | 5.4 | 12× | 64.8 | 2/3 |
| 5.4 | 24× | 129.6 | 3/3 | ||
| 5.4 | 3× | 16.2 | 113 | ||
| 37 | HSV-2 Isolate #14 | 5.4 | 6× | 32.4 | રૂડિયારે તે જેવી સાચના સાચના દિવેલી છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામના |
| 38 | HSV-2 Isolate #15 | 5.4 | 3× | 16.2 | 0/3 |
| 5.4 | 6× | 32.4 | રૂડિયારે તે જેવી સાચના સાચના દિવેલી છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામના | ||
| 39 | HSV-2 Isolate #16 | 5.4 | 3× | 16.2 | 113 |
| 5.4 | 6× | 32.4 | રૂડિયારે તે જેવી સાચના સાચના દિવેલી છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામના | ||
| HSV-2 Isolate #17 | 5.4 | 3× | 16.2 | 113 | |
| 40 | 5.4 | 6× | 32.4 | 113 | |
| 5.4 | 12x | 64.8 | રૂડિયારે તે જેવી સાચના સાચના દિવેલી છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામના | ||
| 41 | HSV-2 Isolate #18 | 5.4 | રૂપ્ | 16.2 | રીકે |
| 42 | HSV-2 Isolate #19 | 5.4 | રૂપ્ | 16.2 | 2/3 |
| 5.4 | 6× | 32.4 | 113 | ||
| 5.4 | 12× | 64.8 | રૂડિયારે તે જેવી સાચના સાચના દિવેલી છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામના | ||
| 5.4 | 3× | 16.2 | 0/3 | ||
| 43 | HSV-2 Isolate #20 | 5.4 | 6× | 32.4 | 113 |
| 5.4 | 12× | 64.8 | 3/3 | ||
| 44 | HSV-2 Isolate #21 | 5.4 | રૂપ્ | 16.2 | રૂડિયારે તે જેવી સાચના સાચના દિવેલી છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામના |
d. Analytical Specificity (Cross-Reactivity)
To determine if there are any organisms that could be potential cross-reactive with the HSV 1&2 ELITE MGB Assay the NCBI BLAST in silico (computer) analysis of the HSV-1 and HSV-2 amplicons was performed. Found sequence homology in both the primer and probe regions was not sufficient for HSV1&2 ELITe MGB Assay to potentially cross-react with the non-HSV organisms.
Potential cross-reactivity of the HSV 1&2 ELITe MGB Assay was then evaluated by testing varied species of organisms that are closely related to HSV or cause similar clinical symptoms or may be present in the anogenital and oral cutaneous and mucocutaneous sites tested by this device. 49 potential cross reactants were evaluated. For each organism, the sample to be tested was prepared from quantified stock diluted to the required concentration using UTM.
The potential cross reactants were tested, the concentrations were evaluated and the results are presented in the following table:
| No. | Potential Cross-Reactants | Tested
Concentration | Qualitative Result
(#Detected/#Total) |
| ----- | --------------------------- | ------------------------- | ------------------------------------------ |
|---|
9
| HSV-1 | HSV-2 | |||
|---|---|---|---|---|
| 1 | Acinetobacter calcoaceticus | 1×106 CFU/mL | 0/3 | 0/3 |
| 2 | Acinetobacter lwoffi | 1×106 CFU/mL | 0/3 | 0/3 |
| 3 | Adenovirus type 2 | 1×105 TCID50/mL | 0/3 | 0/3 |
| 4 | Bacteroides fragilis | 1×106 CFU/mL | 0/3 | 0/3 |
| 5 | Candida albicans | 1×106 CFU/mL | 0/3 | 0/3 |
| 6 | Candida glabrata | 1×106 CFU/mL | 0/3 | 0/3 |
| 7 | Candida guilliermondii | 1×106 CFU/mL | 0/3 | 0/3 |
| 8 | Candida krusei | 1×106 CFU/mL | 0/3 | 0/3 |
| 9 | Candida lustaniae | 1×106 CFU/mL | 0/3 | 0/3 |
| 10 | Candida parapsilosis | 1×106 CFU/mL | 0/3 | 0/3 |
| 11 | Candida tropicalis | 1×106 CFU/mL | 0/3 | 0/3 |
| 12 | Chlamydia trachomatis | 1×106 CFU/mL | 0/3 | 0/3 |
| 13 | Cytomegalovirus | 1×105 TCID50/mL | 0/3 | 0/3 |
| 14 | Enterobacter cloacae | 1×106 CFU/mL | 0/3 | 0/3 |
| 15 | Enterovirus | 1×105 TCID50/mL | 0/3 | 0/3 |
| 16 | Epstein-Barr Virus | 1×105 TCID50/mL | 0/3 | 0/3 |
| 17 | Escherichia coli | 1×106 CFU/mL | 0/3 | 0/3 |
| 18 | Fusobacterium nucleatum | 1×106 CFU/mL | 0/3 | 0/3 |
| 19 | Gardnerella vaginalis | 1×106 CFU/mL | 0/3 | 0/3 |
| 20 | Haemophilus ducreyi | 1×106 CFU/mL | 0/3 | 0/3 |
| 21 | Human Genomic DNA | 500 ng/swab | 0/3 | 0/3 |
| 22 | Human Herpes Virus 6 | 1×105 TCID50/mL | 0/3 | 0/3 |
| 23 | Human Herpes Virus 7 | 1×105 TCID50/mL | 0/3 | 0/3 |
| 24 | Human papilloma virus 16 | 1×105 TCID50/mL | 0/3 | 0/3 |
| 25 | Human papilloma virus 18 | 1×105 TCID50/mL | 0/3 | 0/3 |
| 26 | Herpes Simplex Virus 1 | |||
| (HSV-1), isolate 20, ZMC | 1×105 TCID50/mL | 3/3 | 0/3 | |
| 27 | Herpes Simplex Virus 2 | |||
| (HSV-2), isolate 20. ZMC | 1×105 TCID50/mL | 0/3 | 3/3 | |
| 28 | Klebsiella pneumoniae | 1×106 CFU/mL | 0/3 | 0/3 |
| 29 | Lactobacillus acidophilus | 1×106 CFU/mL | 0/3 | 0/3 |
| 30 | Mobiluncus curtsii | 1×106 CFU/mL | 0/3 | 0/3 |
| 31 | Mobiluncus mulieris | 1×106 CFU/mL | 0/3 | 0/3 |
| 32 | Moraxella catarrhalis | 1×106 CFU/mL | 0/3 | 0/3 |
| 33 | Mycoplasma hominis | 1×106 CFU/mL | 0/3 | 0/3 |
| 34 | Neisseria gonorrhea | 1×106 CFU/mL | 0/3 | 0/3 |
| 35 | Neisseria meningitides | 1×106 CFU/mL | 0/3 | 0/3 |
| 36 | Prevotella melaninogenica | 1×106 CFU/mL | 0/3 | 0/3 |
| 37 | Rubella Virus | 1×105 TCID50/mL | 0/3 | 0/3 |
| 38 | Staphylococcus aureus | |||
| (MSSA) | 1×106 CFU/mL | 0/3 | 0/3 | |
| 39 | Staphylococcus epidermidis | |||
| (MRSE) | 1×106 CFU/mL | 0/3 | 0/3 | |
| 44 | Staphylococcus | |||
| saprophyticus | 1×106 CFU/mL | 0/3 | 0/3 | |
| 41 | Streptococcus mitis | 1×106 CFU/mL | 0/3 | 0/3 |
| 42 | Streptococcus mutans | 1×106 CFU/mL | 0/3 | 0/3 |
| 43 | Streptococcus pneumoniae | 1×106 CFU/mL | 0/3 | 0/3 |
| 44 | Streptococcus pyogenes | 1×106 CFU/mL | 0/3 | 0/3 |
| 45 | Streptococcus salivarius | 1×106 CFU/mL | 0/3 | 0/3 |
| 46 | Toxoplasma gondii | 1×106 CFU/mL | 0/3 | 0/3 |
| 47 | Trichomonas vaginalis | 1×106 CFU/mL | 0/3 | 0/3 |
| 48 | Varicella-Zoster Virus (VZV) | 1×105 TCID50/mL | 0/3 | 0/3 |
| 49 | Chlamydophila pneumoniae | $1 \times 10^6$ CFU/mL | 0/3 | 0/3 |
10
Analytical Specificity (Microbial Interference) e.
The microbial interference was evaluated in the presence of either HSV-1 or HSV-2 spiked at 3×LoD in UTM and the 49 organisms indicated in the table above. Each microorganism was tested either at 1×10° CFU/mL or higher for bacterial isolates, or at 1×10° TCID30/mL or higher for viruses. None of the non-target organisms that are reasonably expected to be found in cutaneous and mucocutaneous swab samples interfered with the detection of HSV-1 or HSV-2 species. The only exception is that amplification of HSV-1 is completely inhibited in the presence of HSV-2 in titers of 1×103 or higher. This observed HSV-2 interference is reported as a limitation in the device package insert.
Competitive Interference of HSV-1 and HSV-2 f.
Competitive interference was studied to evaluate the effects of possible clinically relevant coinfection with both HSV-1 and HSV-2 using HSV 1&2 ELITe MGB Assay.
The study assessed whether a high concentration of one virus in the sample could potentially affect the HSV 1&2 ELITe MGB Assay performance for the other target present at low levels. A low positive sample was contrived at approximately 3×LoD for each target (HSV-1 MacIntyre strain and HSV-2 MS strain), and a baseline Ct was determined for each sample. Each potential concomitant infecting virus was spiked into the low level sample and assaved triplicate. in
Competitive interference of HSV-1 with HSV-2 was observed at 1×10°, 1×10°, 1×10° TCIDss/mLHSV-2 level. This observed interference information is included as a limitation in the device package insert.
No competitive interference of HSV-2 with HSV-1 at any level was observed. The results of the testing are shown in the table below.
| Baseline (Low Level) | | Competitive Interferent
(High Concentration) | | Qualitative Results
(#Detected/#Total) | |
|----------------------|------------------------------|-------------------------------------------------|------------------------------|-------------------------------------------|-------|
| Strain | Concentration
(TCID50/mL) | Strain | Concentration
(TCID50/mL) | HSV-1 | HSV-2 |
| HSV-1 MacIntyre | 177 | HSV-2 MS | 100000 | 0/3 | 3/3 |
| HSV-1 MacIntyre | 177 | HSV-2 MS | 10000 | 1/3 | 3/3 |
| HSV-1 MacIntyre | 177 | HSV-2 MS | 1000 | 1/3 | 3/3 |
| HSV-1 MacIntyre | 177 | HSV-2 MS | 100 | 3/3 | 3/3 |
| HSV-1 MacIntyre | 177 | HSV-2 MS | 0 | 3/3 | 0/3 |
| HSV-2 MS | 16.2 | HSV-1 MacIntyre | 100000 | 3/3 | 3/3 |
| HSV-2 MS | 16.2 | HSV-1 MacIntyre | 10000 | 3/3 | 3/3 |
| HSV-2 MS | 16.2 | HSV-1 MacIntyre | 1000 | 3/3 | 3/3 |
| HSV-2 MS | 16.2 | HSV-1 MacIntyre | 100 | 3/3 | 3/3 |
| HSV-2 MS | 16.2 | HSV-1 MacIntyre | 0 | 0/3 | 3/3 |
Competitive Interference of HSV-1 and HSV-2 targets at unequal concentrations
Additionally, in a separate study both strains were tested at similar or equal concentrations of 3×LoD, 1×103 and 1×105, and no competitive interference was observed.
| HSV-1 Concentration | HSV-2 Concentration | Qualitative Results
(#Detected/#Total) | Quantitative Results (%CV) |
| --------------------- | --------------------- | ------------------------------------------- | ---------------------------- |
|---|
11
| Strain | TCID50/mL | Strain | TCID50/mL | HSV-1 | HSV-2 | HSV-1 | HSV-2 |
|---|---|---|---|---|---|---|---|
| HSV-1 | |||||||
| MacIntyre | $1\times10^5$ | HSV-2 MS | $1\times10^5$ | 5/5 | 5/5 | 3.02 % | 1.64 % |
| HSV-1 | |||||||
| MacIntyre | $1\times10^3$ | HSV-2 MS | $1\times10^3$ | 5/5 | 5/5 | 1.09 % | 2.95 % |
| HSV-1 | |||||||
| MacIntyre | 177 (3×LoD) | HSV-2 MS | 16.2 (3×LoD) | 5/5 | 5/5 | 1.74 % | 1.88 % |
g. Interfering Substances
The performance of the HSV 1&2 ELITe MGB Assay was evaluated with potentially interfering substances that could be encountered in lesion swab specimens obtained from cutaneous and mucocutaneous locations. A total of 33 substances were individually spiked into negative UTM matrix containing one of each HSV-1 and HSV-2 isolates at 3X LoD level and tested in triplicate with the HSV 1&2 ELITe MGB Assay. There were no invalid result calls. No interference was observed (see table below).
| Potential Interferent | Interferent
Concentration | #Detected/#Total
HSV-1 | #Detected/#Total
HSV-2 | IC |
|----------------------------------------|------------------------------|---------------------------|---------------------------|-----|
| Whole blood with EDTA | 5% v/v | 0/3 | 0/3 | 3/3 |
| Buffy coat | 5% v/v | 0/3 | 0/3 | 3/3 |
| Acyclovir | 2.5 mg/mL | 0/3 | 0/3 | 3/3 |
| Albumin | 5 mg/mL | 0/3 | 0/3 | 3/3 |
| Casein | 7 mg/mL | 0/3 | 0/3 | 3/3 |
| Female urine | 10% v/v | 0/3 | 0/3 | 3/3 |
| Male urine | 10% v/v | 0/3 | 0/3 | 3/3 |
| K-Y Brand jelly | 5% w/v | 0/3 | 0/3 | 3/3 |
| Douche | 10% v/v | 0/3 | 0/3 | 3/3 |
| Spermicide | 5% w/v | 0/3 | 0/3 | 3/3 |
| Yeast-Gard | 1% w/v | 0/3 | 0/3 | 3/3 |
| Monistat 1 | 5% w/v | 0/3 | 0/3 | 3/3 |
| Monistat 3 | 5% w/v | 0/3 | 0/3 | 3/3 |
| Vagisil Cream | 1% w/v | 0/3 | 0/3 | 3/3 |
| Tioconazole 1 | 5% w/v | 0/3 | 0/3 | 3/3 |
| Rite Aid Feminine Wash, Sensitive Skin | 10% v/v | 0/3 | 0/3 | 3/3 |
| Clotrimazole-7 vaginal cream | 1% w/v | 0/3 | 0/3 | 3/3 |
| Oral Analgesic Gel | 5% w/v | 0/3 | 0/3 | 3/3 |
| Listerine antiseptic mouthwash | 10% v/v | 0/3 | 0/3 | 3/3 |
| Abreva | 10% v/v | 0/3 | 0/3 | 3/3 |
| Carmex lip balm | 1% w/v | 0/3 | 0/3 | 3/3 |
| Releev cold sore treatment | 1% v/v | 0/3 | 0/3 | 3/3 |
| Lip Clear lysine | 1% w/v | 0/3 | 0/3 | 3/3 |
| Toothpaste | 5% w/v | 0/3 | 0/3 | 3/3 |
| Acetaminophen | 5 mg/mL | 0/3 | 0/3 | 3/3 |
| Wal-Finate | 5 mg/mL | 0/3 | 0/3 | 3/3 |
| Cold-Eeze | 7% w/v | 0/3 | 0/3 | 3/3 |
| Non-GMO Corn Starch | 1.25 mg/mL | 0/3 | 0/3 | 3/3 |
| Zinc Oxide Ointment | 7% w/v | 0/3 | 0/3 | 3/3 |
| Cough DM | 10mg/mL | 0/3 | 0/3 | 3/3 |
| Lanacane Max Strength anti-itch cream | 7% w/v | 0/3 | 0/3 | 3/3 |
| Seminal fluid | 7% v/v | 0/3 | 0/3 | 3/3 |
| Foscarnet sodium | 5% v/v | 0/3 | 0/3 | 3/3 |
12
h. Carry-Over Contamination
The sample-to-sample carry-over from positive samples into the negative samples for the HSV 1&2 ELITe MGB Assay was studied by performing 5 integrated checkerboard runs (high positive for HSV-1 at 2.5×10′ TCIDso/mL concentration and negative samples (UTM) interspersed), which were compared to the overall contamination level ("background noise") of 2 negative sample runs. One operator ran 7 runs (5 checkerboard runs and 2 complete neqative runs). The High positive sample concentration in this study were high enough to exceed 95% or more of the concentration levels obtained from specimens of infected patients in the intended use population. No carry-over and cross contamination was observed. Overall percent agreement was 100% for positive and negative samples.
| Run description | Positive Samples
Neg | % Neg. | Negative Samples
Pos. | % Pos. |
|----------------------|---------------------------|--------|----------------------------|--------|
| Run #1, BLANK | 0 / 0 | NA | 0 / 10 | 0 % |
| Run #2, Checkerboard | 0 / 5 | 0 % | 0 / 6 | 0 % |
| Run #3, Checkerboard | 0 / 6 | 0 % | 0 / 6 | 0 % |
| Run #4, BLANK | 0 / 0 | NA | 0 / 10 | 0 % |
| Run #5, Checkerboard | 0 / 6 | 0 % | 0 / 6 | 0 % |
| Run #6, Checkerboard | 0 / 6 | 0 % | 0 / 6 | 0 % |
| Run #7, Checkerboard | 0 / 6 | 0 % | 0 / 6 | 0 % |
| All runs | 0 / 29 | 0 % | 0 / 50 | 0 % |
Carry-Over and Cross-Contamination Results
Sample Stability i.
This study assessed both sample stability and sample freeze-thaw stability. The samples for the stability evaluation were prepared by spiking both the HSV-1 and HSV-2 vendor quantitated viral stocks (HSV-1 MacIntyre strain and HSV-2 MS strain) in UTM, M4, M4RT, M5 and M6 media.
Each stability sample set consisted of:
- 5 replicates spiked at 3×LoD. .
- 5 replicates spiked at 1×103 TCIDso/mL, and ●
- 5 replicates spiked at 1×10° TCIDso/mL (15 replicates total for each sample set). .
The stability of each sample set was assessed by sample incubation at +4°C for 1 week. All HSV-1 and HSV-2 samples were confirmed to be stable in UTM, M4, M4RT, M5 and M6 media for 1 week at +4°C.
The storage conditions were also validated by re-testing previously analyzed clinical samples that were stored in a -80°C freezer (≤ -70°C) for minimum of 4 months. Sample concentrations covered HSV clinical range. Ten HSV-1 or HSV-2 positive samples were tested for each media (except M6 for which only 7 HSV-positive samples were available). Positivity of all samples was confirmed after 4 month storage in a -80°C freezer (≤ -70°C).
A freeze thaw study was performed using 5 sample sets prepared as in the above using UTM, M4. M4RT, M5 and M6 media. All samples were subjected to 3 freeze-thaw cycles. All the samples were tested with the HSV 1&2 ELITe MGB Assay on the ELITe InGenius. The data obtained show that HSV-1 and HSV-2 viruses are stable after 3 freeze-thaw cycles in UTM, M4, M4RT, M5 and M6 media.
Matrix Comparison Study -
Since all analytical studies were conducted in the UTM (Universal Transport Media) and clinical studies were conducted using UTM, M4, M4RT, M5 and M6 media, the matrix comparison study was performed. The matrix comparison study was conducted using contrived sample panel made by spiking either HSV-1 or HSV-2 quantitated viral organisms
13
into each of the recommended media: UTM, M4, M4RT, M5 and M6. Each sample set consisted of 3 replicates spiked at 3×LoD, 3 replicates spiked at 1×10° TCIDsomL, and 3 replicates spiked at 1×10 TCID50/mL (9 replicates total for each sample set). Each sample was processed on the InGenius using the HSV 1&2 ELITe MGB Assay. All replicates in all media were detected and showed comparable results.
| Target/
Channel | Sample
Titer
TCID50/mL | UTM,
Avg CT | Sample Matrix | | | | All
Media
Avg CT | All
Media
StDev | All
Media
%CV |
|------------------------|------------------------------|----------------|---------------|-----------------|---------------|---------------|------------------------|-----------------------|---------------------|
| | | | M4,
Avg CT | M4RT,
Avg CT | M5,
Avg CT | M6,
Avg CT | | | |
| HSV-2
CH1,
FAM | 1.00E+05 | 27.15 | 26.76 | 26.42 | 26.82 | 27.23 | 26.88 | 0.33 | 1.21% |
| | 1.00E+03 | 33.86 | 33.59 | 33.76 | 33.51 | 34.15 | 33.77 | 0.25 | 0.74% |
| | 3×LoD | 36.32 | 35.56 | 35.96 | 35.54 | 36.14 | 35.91 | 0.35 | 0.97% |
| HSV-1
CH4,
AP593 | 1.00E+05 | 22.02 | 21.13 | 20.82 | 20.77 | 20.63 | 21.08 | 0.56 | 2.66% |
| | 1.00E+03 | 28.01 | 28.47 | 27.97 | 28.58 | 26.72 | 27.95 | 0.74 | 2.64% |
| | 3×LoD | 35.84 | 36.07 | 37.02 | 35.43 | 34.69 | 35.81 | 0.86 | 2.39% |
All tested media showed comparable performance.
k. Reagent Stability
The stability of the HSV 1&2 ELITe MGB Assay was evaluated using several different methods.
Real-time Stability
A real-time stability was completed over a 12 month time period and the data support a shelf-life claim of 10 months for the HSV 1&2 ELITe MGB Assay.
Freeze-thaw Stability
Three lots of HSV 1&2 ELITe MGB Assay reagents were thawed, opened and held for a period of one hour at 30°C and then re-frozen by re-capping and replacing in ≤ -20°C (nominal) freezer. This cycle was repeated 8 times (once per day). Results show that the HSV 1&2 ELITe MGB Assay is stable when subjected to 8 freeze-thaw cycles.
11. Performance Characteristics
a. Reproducibility
The reproducibility of the HSV 1&2 ELITe MGB Assay was evaluated in a multi-site investigation using contrived clinical samples. HSV test panels were prepared by spiking HSV-1 (Maclntyre strain) or HSV-2 (MS strain) virus into UTM media at the concentrations of 630 reproducibility samples (minimum 210 per testing site). Lot-to-Lot variability was assessed only at EGI MDx using 3 lots of HSV 1&2 ELITe MGB Assay. Controls were run daily and were included in the first run of the day.
% Agreement, average Cts and %CV for each panel member and per each site are presented in the table below.
| Site - 1 | Site - 2 | Site - 3 | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| % | % | % | % | |||||||||
| Agree- | Agree- | Agree- | Agree- | |||||||||
| ment with | ment with | ment with | ment with | |||||||||
| Expected | Avg. | Total | Expected | Avg. | Total | Expected | Avg. | Total | Expected | |||
| Target | Sample | Results | Ct | %CV | Results | Ct | %CV | Results | Ct | %CV | Results | 95% Cl |
| HSV-1 | 100.0% | 100.0% | 100.0% | 100.0% | 95.9 to | |||||||
| Low Pos | (30/30) | 38.9 | 1.70% | (30/30) | 38.3 | 2.10% | (30/30) | 38 | 2.00% | (90/90) | 100.0% | |
| HSV-1 | 100.0% | 100.0% | 100.0% | 100.0% | 95.9 to | |||||||
| Mod Pos | (30/30) | 36.4 | 1.30% | (30/30) | 35.5 | 5.20% | (30/30) | 35.6 | 1.50% | (90/90) | 100.0% | |
| HSV-2 | 100.0% | 100.0% | 100.0% | 100.0% | 95.6 to | |||||||
| Low Pos | (30/30) a | NA | NA | (29/29) a | NA | NA | (30/30) a | NA | NA | (89/89) | 100.0% | |
| HSV-1 | HSV-2 | 100.0% | 100.0% | 100.0% | 100.0% | 95.9 to | ||||||
| Result | Mod Pos | (30/30) ª | NA | NA | (30/30) ª | NA | NA | (30/30) a | NA | NA | (90/90) | 100.0% |
| HSV | 100.0% | 100.0% | 100.0% | 100.0% | 97.3 to | |||||||
| (60/60) | NA | NA | (38/38) a | 41.4 | 2.50% | (40/40) | NA | NA | (138/138) | 100.0% | ||
| Neg | 100.0% | |||||||||||
| Pos | 100.0% | 27.5 | 1.30% | 100.0% | ||||||||
| (515) | 27.5 | 1.20% | 100.0% | |||||||||
| (515) | 27 | 0.80% | 91.2 to | |||||||||
| Control | (30/30) | 100.0% | (40/40) | 100.0% | ||||||||
| Total | (210/210) | 100.0% | 100.0% | 100.0% | 98.2 to | |||||||
| Agreement | (162/162) | (165/165) | 5371537) | 100.0% | ||||||||
| 4 Expected Results of HSV-2 Low Positive, HSV-2 Moderate Positive samples are "Negative" for HSV-1. | ||||||||||||
| Site - 1 | Site - 2 | Site - 3 | ||||||||||
| % | % | % | % | |||||||||
| Agree- | Agree- | Agree- | Agree- | |||||||||
| ment with | ment with | ment with | ment with | |||||||||
| Expected | Avg. | Total | Expected | Avg. | Total | Expected | Avg. | Total | Expected | |||
| Target | Sample | Results | Ct | %CV | Results | Ct | %CV | Results | Ct | %CV | Results | 95% Cl |
| HSV-1 | 100.0% | NA | NA | 100.0% | NA | NA | 100.0% | NA | NA | 100.0% | 95.9 to | |
| Low Pos | (30/30) b | (30/30) 6 | (30/30) b | (90/90) | 100.0% | |||||||
| HSV-1 | 100.0% | NA | NA | 100.0% | NA | NA | 100.0% | NA | NA | 100.0% | 95.9 to | |
| Mod Pos | (30/30) P | (30/30) P | (30/30) P | (90/90) | 100.0% | |||||||
| HSV-2 | 100.0% | 36.8 | 3.10% | 100.0% | 37.8 | 2.30% | 100.0% | 36.6 | 1.90% | 100.0% | 95.9 to | |
| Low Pos | (30/30) | (29/29) | (30/30) | (89/89) | 100.0% | |||||||
| HSV-2 | HSV-2 | 100.0% | 35.2 | 1.30% | 100.0% | 35.95 | 1.60% | 100.0% | 34.6 | 2.30% | 100.0% | 95.9 to |
| Result | Mod Pos | (30/30) | (30/30) | (30/30) | (90/90) | 100.0% | ||||||
| HSV | 100.0% | 100.0% | 100.0% | 100.0% | 95.9 to | |||||||
| Neg | (60/60) 6 | NA | NA | (38/38) 6 | NA | NA | (40/40) 6 | NA | NA | (138/138) | 100.0% | |
| Pos | 100.0% | 100.0% | 100.0% | 100.0% | 95.9 to | |||||||
| Control | (30/30) | 27 | 1.30% | (5/5) | 27.4 | 1.50% | (5/5) | 26.8 | 1.40% | (40/40) | 100.0% | |
| Total | 100.0% | 100.0% | 100.0% | 100.0% | 95.9 to | |||||||
| Agreement | (210/210) | (162/162) | (165/165) | 5371537 | 100.0% | |||||||
| P Expected Results of HSV-1 Low Positive, HSV-1 Moderate Positive samples are "Negative" for HSV-2. | ||||||||||||
| Site - 1 | Site - 2 | Site - 3 | ||||||||||
| % | % | % | % | |||||||||
| Agree- | Agree- | Agree- | Agree- | |||||||||
| ment with | ment with | ment with | ment with | |||||||||
| Expected | Avg | Total | Expected | Avg. | Total | Expected | Avg. | Total | Expected | |||
| Target | Sample | Results | Ct | %CV | Results | Ct | %CV | Results | Ct | %CV | Results | 95% Cl |
| HSV-1 | 100.0% | 100.0% | 100.0% | 100.0% | 95.9 to | |||||||
| Low Pos | (30/30) | 30.4 | 3.80% | (30/30) | 30.3 | 2.00% | (30/30) | 30.2 | 1.70% | (90/90) | 100.0% | |
| HSV-1 | 100.0% | 100.0% | 100.0% | 100.0% | 95.9 to | |||||||
| IC | Mod Pos | (30/30) | 30.2 | 2.30% | (30/30) | 30.4 | 2.80% | (30/30) | 30.1 | 0.90% | (90/90) | 100.0% |
| Result | HSV-2 | 100.0% | 100.0% | 100.0% | 100.0% | 95.9 to | ||||||
| Low Pos | (30/30) | 29.9 | 0.50% | (29/29) | 30.4 | 2.20% | (30/30) | 30.2 | 0.60% | (89/89) | 100.0% | |
| HSV-2 | 100.0% | 100.0% | 100.0% | 100.0% | 95.9 to | |||||||
| Mod Pos | (30/30) | 29.7 | 0.80% | (30/30) | 30.4 | 1.20% | (30/30) | 30.1 | 0.60% | (90/90) | 100.0% | |
| HSV | ||||||||||||
| Neg | 100.0% | |||||||||||
| (60/60) | 30.2 | 1.10% | 100.0% | |||||||||
| (40/40) | 30.2 | 1.90% | 100.0% | |||||||||
| (38/38) | 30.1 | 0.90% | 100.0% | |||||||||
| (138/138) | 97.3 to | |||||||||||
| 100.0% | ||||||||||||
| Pos | ||||||||||||
| Control | 100.0% | |||||||||||
| (30/30) | 29.3 | 1.40% | 100.0% | |||||||||
| (5/5) | 30.2 | 2.10% | 100.0% | |||||||||
| (5/5) | 29.4 | 0.90% | 100.0% | |||||||||
| (40/40) | 91.2 to | |||||||||||
| 100.0% | ||||||||||||
| Total | ||||||||||||
| Agreement | 100.0% | |||||||||||
| (210/210) | 100.0% | |||||||||||
| (164/164) | 100.0% | |||||||||||
| (163/163) | 100.0% | |||||||||||
| (537/537) | 98.2 to | |||||||||||
| 100.0% |
15
The highest HSV 1&2 ELITe MGB Assay Site-to-Site variability (as measured by %CV based on Ct values) is 2.19%; the highest Lot is 0.23%, and the highest Operator variability is 0.93% for Moderate Positive panel members.
b. Clinical Performance
To evaluate the clinical performance of the HSV 1&2 ELITe MGB Assay, device performance was compared to a composite reference method. It consisted of an FDA cleared assay and a validated HSV 1&2 PCR followed by bi-directional sequencing of gel electrophoresis-positive samples). Validated HSV 1&2 PCR targeted genomic regions distinct from the HSV 1&2 ELITe MGB Assay. A positive result by the composite reference method is defined as a positive by the FDA cleared PCR or the validated sequencing. Two negative results are needed to confirm a negative)
A total of 1,174 left-over prospectively collected archived swab samples from cutaneous (546) and mucocutaneous (628) lesions from symptomatic patients were collected and evaluated in the study.
The samples were tested with HSV 1&2 ELITe MGB Assay and the Composite Reference Method. Out of the 1,174 tested samples were found invalid by the ELITe MGB Assay and were excluded from the performance analysis tables.
Out of the 1172 remaining samples 1 additional invalid sample result for HSV1 and 2 additional invalid sample results for HSV2 by the composite reference method were removed from the performance analysis tables.
Therefore for HSV1, 1171 samples analyzed and for HSV2 1170 samples were analyzed.
HSV-1 Positive/Neqative Percent Agreements (PPA/NPA) - Summary of the Results:
The PPA/NPA performance of HSV 1&2 ELITe MGB Assay when compared to the Composite Reference Method in detection of HSV-1 DNA in cutaneous and mucocutaneous lesions is summarized in the table below:
| Summary of HSV-1 Results for Valid Cutaneous Lesion Samples (N=545) | |||
|---|---|---|---|
| HSV 1&2 ELITe MGB Kit | Composite reference method | ||
| Positive | Negative | Total | |
| Positive | 78 | 7 | 85 |
| Negative | 1 | 459 | 460 |
| Total | 79 | 466 | 545 |
| 95% CI | |||
| PPA | 98.7% | 93.2-99.8% | |
| NPA | 98.5% | 96.9-99.3% |
| Summary of HSV-1 Results for Valid Mucocutaneous Lesion Samples (N=626) | |||
|---|---|---|---|
| HSV 1&2 ELITE MGB Kit | Composite reference method | ||
| Positive | Negative | Total | |
| Positive | 126 | 12 | 138 |
| Negative | 1 | 487 | 488 |
| Total | 127 | 499 | 626 |
| 95% CI | |||
| PPA | 99.2% | 95.7-99.9% | |
| NPA | 97.6% | 95.8-98.6% |
16
HSV-2 Positive/Negative Percent Agreements (PPA/NPA) - Summary of the Results:
The PPA/NPA performance of HSV 1&2 ELITe MGB Assay when compared to the Composite Reference Method in detection of HSV-2 DNA in cutaneous and mucocutaneous lesions is summarized in the table below:
| Summary of HSV-2 Results for Valid Cutaneous Lesion Samples (N=545) | |||
|---|---|---|---|
| HSV 1&2 ELITe MGB Kit | Composite reference method | ||
| Positive | Negative | Total | |
| Positive | 125 | 6 | 131 |
| Negative | 5 | 409 | 414 |
| Total | 130 | 415 | 545 |
| 95% CI | |||
| PPA | 96.2% | 91.3-98.3% | |
| NPA | 98.6% | 96.9-99.3% |
| Summary of HSV-2 Results for Valid Mucocutaneous Lesion Samples (N=625) | |||
|---|---|---|---|
| HSV 1&2 ELITe MGB Kit | Composite reference method | ||
| Positive | Negative | Total | |
| Positive | 164 | 8 | 172 |
| Negative | 4 | 449 | 453 |
| Total | 168 | 457 | 625 |
| 95% CI | |||
| PPA | 97.6% | 94.0-99.1% | |
| NPA | 98.2% | 96.6-99.1% |
Results: Expected values/Reference Range ﻦ
The observed expected values for HSV-1 and HSV-2 in the study population using the HSV 1&2 ELITe MGB Assay were calculated for cutaneous and mucocutaneous specimens and is summarized for the combined sample set per age group, by gender and by specimen source in the tables below. A total number of 6 dual positives for HSV1 and HSV2 detected by the ELITe MGB Assay and one of the samples was confirmed positive by the composite reference method.
| Gender | Age Group | Total | HSV 1&2 ELITe MGB
Assay HSV-1 results | | HSV 1&2 ELITe MGB
Assay HSV-2 results | |
|--------|-----------|-------|------------------------------------------|------------|------------------------------------------|------------|
| | | | Positive | Prevalence | Positive | Prevalence |
| Female |