cobas HSV 1 and 2 Test
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 |
Indications for 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)
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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY
A. 510(k) Number:
K150617
B. Purpose for Submission:
Clearance of New Device
C. Measurand:
Target DNA sequences from Herpes Simplex Virus type 1 (HSV-1) and Herpes Simplex Virus type 2 (HSV-2)
D. Type of Test:
An *in vitro* molecular diagnostic test for the qualitative detection and differentiation of HSV-1 and HSV-2 DNA in clinician-collected, external anogenital lesion specimens from symptomatic male and female patients.
E. Applicant:
Roche Molecular Systems, Inc.
F. Proprietary and Established Names:
cobas® HSV 1 and 2 Test
G. Regulatory Information:
1. Regulation section: 21 CFR 866.3305
2. Classification: Class II
3. Product code: OQO
4. Panel: Microbiology (83)
H. Intended Use:
1. Intended use(s):
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)
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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.
2. **Indication(s) for use:**
Same as Intended Use
3. **Special conditions for use statement(s):**
For prescription use only
4. **Special instrument requirements:**
cobas® 4800 System
I. **Device Description:**
The cobas® HSV 1 and 2 Test on the cobas® 4800 system is a real-time polymerase chain reaction (PCR) for the direct detection and differentiation of HSV-1 and HSV-2 DNA in clinician-collected, external anogenital lesion specimens, collected in MSwab Collection, Transport and Preservation System from symptomatic patients.
The cobas® HSV 1 and HSV 2 Test is comprised of 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 specimens are collected and stored in the MSwab Collection, Transport and Preservation System for the cobas® HSV 1 and HSV 2 Test. The cobas® HSV 1 and HSV 2 Test is performed using the following reagent kits:
1. cobas® 4800 HSV 1 and HSV 2 Amplification/Detection Kit
2. cobas® 4800 HSV 1 and HSV 2 Controls and Cofactor Kit
3. cobas® 4800 System Wash Buffer Kit
4. cobas® 4800 System Lysis Kit 1
5. cobas® 4800 System Internal Control Kit 1
6. cobas® 4800 System Sample Preparation Kit
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J. Substantial Equivalence Information:
1. Predicate device name(s):
BD ProbeTec™ Herpes Simplex Viruses (HSV 1 & 2) Q⁺ Amplified DNA Assays
Reference Method:
A composite reference method which includes a culture method, namely, the ELVIS® HSV ID/Typing Test System (Diagnostic Hybrids, Inc. (K971662)) and a validated PCR followed by bidirectional sequencing for clinical evaluation.
The clinical performance of the cobas® HSV 1/2 Test was also compared to the culture method alone using the ELVIS® HSV ID/Typing Test System (Diagnostic Hybrids, Inc. (K971662)).
2. Predicate 510(k) number(s):
K103798
3. Comparison with predicate:
| Similarities | | |
| --- | --- | --- |
| Characteristic | BD ProbeTec™ Herpes Simplex Viruses (HSV 1 & 2) Q⁺ 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 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. | 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 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 |
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| | 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. | 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 |
K. Standard/Guidance Document Referenced (if applicable): N/A
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L. Test Principle:
Target Selection
The cobas® HSV 1 and 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. The particles are washed and the bound nucleic acids are 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® z 480 Analyzer. The Master Mix reagent contains primer pairs and probes for HSV-1, HSV-2 and Internal Control 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 is subsequently 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® z 480 analyzer. The signal is interpreted by the cobas® 4800 System Software and reported as final results.
M. Performance Characteristics (if/when applicable):
1. Analytical performance:
a. Reproducibility:
The reproducibility of the cobas® HSV 1 and 2 Test on the cobas® 4800 System was established at three sites using contrived clinical samples evaluated across lot, site/instrument, operator, day, and run. HSV test panels were prepared by spiking HSV-1 (MacIntyre strain) and/or HSV-2 (G strain) 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
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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 216 tests per panel member.
## HSV-1 reproducibility results
The table below summarizes the percent agreement (two-sided 95% exact Confidence Interval (CI) for the negative panel member and HSV-1 positive panel members.
### HSV-1 Percent agreement by panel member
| | | HSV-1 | |
| --- | --- | --- | --- |
| Panel Member | Number of Valid Test Results | Agreement (n/N) | 95% CI^{a} |
| Negative^{b} | 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%) |
a 95% CI = Two-sided 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%).
c 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.
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The table below presents the percent agreement (negative or positive) by lot, site/instrument, operator, and day for HSV1 test results for each panel.
HSV-1 Percent agreement by panel member for lot, site/instrument, operator, and day
| | | | | 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 |
| | | | | 2 | 100.0 (108/108) | 2 | 100.0 (72/72) | 2 | 100.0 (36/36) | 2 |
| | | | | | | 3 | 100.0 (72/72) | 3 | 100.0 (36/36) | 3 |
| | | | | | | | | 4 | 100.0 (36/36) | 4 |
| | | | | | | | | 5 | 100.0 (36/36) | 5 |
| | | | | | | | | 6 | 100.0 (36/36) | 6 |
| | | | | | | | | | | |
| 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 |
| | | | | 2 | 66.7 (72/108) | 2 | 68.1 (49/72) | 2 | 55.6 (20/36) | 2 |
| | | | | | | 3 | 65.3 (47/72) | 3 | 63.9 (23/36) | 3 |
| | | | | | | | | 4 | 72.2 (26/36) | 4 |
| | | | | | | | | 5 | 63.9 (23/36) | 5 |
| | | | | | | | | 6 | 66.7 (24/36) | 6 |
| | | | | | | | | | | |
| 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 |
| | | | | 2 | 100.0 (108/108) | 2 | 100.0 (72/72) | 2 | 100.0 (36/36) | 2 |
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| | | | | Percent Agreement (n/N)* | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Panel Member | Ct | | | Lot | Site/Inst. | Operator | Day | | | |
| | Mean | SD | CV % | | | | | | | |
| | | | | | | 3 | 100.0 (72/72) | 3 | 100.0 (36/36) | 3 |
| | | | | | | | | 4 | 100.0 (36/36) | 4 |
| | | | | | | | | 5 | 100.0 (36/36) | 5 |
| | | | | | | | | 6 | 100.0 (36/36) | 6 |
| | | | | | | | | | | |
| 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 |
| | | | | 2 | 100.0 (108/108) | 2 | 100.0 (72/72) | 2 | 100.0 (36/36) | 2 |
| | | | | | | 3 | 100.0 (72/72) | 3 | 100.0 (36/36) | 3 |
| | | | | | | | | 4 | 100.0 (36/36) | 4 |
| | | | | | | | | 5 | 100.0 (36/36) | 5 |
| | | | | | | | | 6 | 100.0 (36/36) | 6 |
| | | | | | | | | | | |
| 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 |
| | | | | 2 | 100.0 (108/108) | 2 | 100.0 (72/72) | 2 | 100.0 (36/36) | 2 |
| | | | | | | 3 | 100.0 (72/72) | 3 | 100.0 (36/36) | 3 |
| | | | | | | | | 4 | 100.0 (36/36) | 4 |
| | | | | | | | | 5 | 100.0 (36/36) | 5 |
| | | | | | | | | 6 | 100.0 (36/36) | 6 |
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| | | | | Percent Agreement (n/N)* | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Panel Member | Ct | | | Lot | Site/Inst. | Operator | Day | | | |
| | Mean | SD | CV % | | | | | | | |
| 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 |
| | | | | 2 | 100.0 (108/108) | 2 | 100.0 (72/72) | 2 | 100.0 (36/36) | 2 |
| | | | | | | 3 | 100.0 (72/72) | 3 | 100.0 (36/36) | 3 |
| | | | | | | | | 4 | 100.0 (36/36) | 4 |
| | | | | | | | | 5 | 100.0 (36/36) | 5 |
| | | | | | | | | 6 | 100.0 (36/36) | 6 |
* 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.
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The table below 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%.
## HSV-1 Overall mean, standard deviations, and coefficients of variation (%) for Ct values from valid results for positive panel members
| | 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 |
| 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 |
| 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 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 |
| 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 |
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% CI: 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
The table below summarizes the percent agreement (two-sided 95% exact CI) for the negative panel member and HSV-2 positive panel members.
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HSV-2 Percent agreement by panel member
| | | HSV-2 | |
| --- | --- | --- | --- |
| Panel Member | Number of Valid Test Results | Agreement (n/N) | 95% CI^{a} |
| Negative^{b} | 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%) |
a 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%).
c 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 the at analyte or when the negative panel member has a valid result of negative for both analytes.
CI = confidence interval; LOD = limit of detection.
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The table below presents the percent agreement (negative and positive) by lot, site/instrument, operator, and day for HSV-2 test results for each panel member.
HSV-2 Percent agreement by panel member for lot, site/instrument, operator, and day
| | | | | 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 |
| | | | | 2 | 100.0 (108/108) | 2 | 100.0 (72/72) | 2 | 100.0 (36/36) | 2 |
| | | | | | | 3 | 100.0 (72/72) | 3 | 100.0 (36/36) | 3 |
| | | | | | | | | 4 | 100.0 (36/36) | 4 |
| | | | | | | | | 5 | 100.0 (36/36) | 5 |
| | | | | | | | | 6 | 100.0 (36/36) | 6 |
| | | | | | | | | | | |
| Below LOD (HSV-1/HSV-2) | 40.3 | 0.89 | 2.2 | 1 | 51.9 (56/108) | 1 | 65.3 (47/72) | 1 | 58.3 (21/36) | 1 |
| | | | | 2 | 61.1 (66/108) | 2 | 50.0 (36/72) | 2 | 72.2 (26/36) | 2 |
| | | | | | | 3 | 54.2 (39/72) | 3 | 58.3 (21/36) | 3 |
| | | | | | | | | 4 | 38.9 (14/36) | 4 |
| | | | | | | | | 5 | 61.1 (22/36) | 5 |
| | | | | | | | | 6 | 50.0 (18/36) | 6 |
| | | | | | | | | | | |
| 1 x LOD (HSV-1) | N/A | N/A | N/A | 1 | 100.0 (108/108) | 1 | 100.0 (72/72) | 1 | 100.0 (36/36) | 1 |
| | | | | 2 | 100.0 (108/108) | 2 | 100.0 (72/72) | 2 | 100.0 (36/36) | 2 |
| | | | | | | 3 | 100.0 (72/72) | 3 | 100.0 (36/36) | 3 |
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| | | | | Percent Agreement (n/N)* | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Panel Member | Ct | | | Lot | Site/Inst. | Operator | Day | | | |
| | Mean | SD | CV % | | | | | | | |
| | | | | | | | 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-2) | 39.0 | 0.92 | 2.3 | 1 | 100.0 (108/108) | 1 | 100.0 (72/72) | 1 | 100.0 (36/36) | 1 |
| | | | | 2 | 100.0 (108/108) | 2 | 100.0 (72/72) | 2 | 100.0 (36/36) | 2 |
| | | | | | | 3 | 100.0 (72/72) | 3 | 100.0 (36/36) | 3 |
| | | | | | | | | 4 | 100.0 (36/36) | 4 |
| | | | | | | | | 5 | 100.0 (36/36) | 5 |
| | | | | | | | | 6 | 100.0 (36/36) | 6 |
| | | | | | | | | | | |
| 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 |
| | | | | 2 | 100.0 (108/108) | 2 | 100.0 (72/72) | 2 | 100.0 (36/36) | 2 |
| | | | | | | 3 | 100.0 (72/72) | 3 | 100.0 (36/36) | 3 |
| | | | | | | | | 4 | 100.0 (36/36) | 4 |
| | | | | | | | | 5 | 100.0 (36/36) | 5 |
| | | | | | | | | 6 | 100.0 (36/36) | 6 |
| | | | | | | | | | | |
| 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 |
{13}
| | | | | Percent Agreement (n/N)* | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Panel Member | Ct | | | Lot | Site/Inst. | Operator | Day | | | | |
| | Mean | SD | CV % | | | | | | | | |
| | | | | 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 = instrument; LOD = limit of detection; N/A = not applicable; SD = standard deviation.
HSV-2 Overall mean, standard deviations, and coefficients of variation (%) for Ct values from valid results for positive panel members
| | Standard Deviation and Percent Coefficient 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 % | CV % |
| Below LOD (HSV-1/HSV-2) | 122 | 40.3 | 0.08 | 0.2 | 0.36 | 0.9 | 0.00 | 0.0 | 0.26 | 0.7 | 0.76 | 1.9 | 0.89 |
| 1 x LOD (HSV-2) | 216 | 39.0 | 0.03 | 0.1 | 0.68 | 1.7 | 0.00 | 0.0 | 0.31 | 0.8 | 0.53 | 1.4 | 0.92 |
| 3 x LOD (HSV-1)/1 x LOD (HSV-2) | 216 | 38.8 | 0.00 | 0.0 | 0.64 | 1.7 | 0.00 | 0.0 | 0.21 | 0.5 | 0.54 | 1.4 | 0.86 |
| 1 x LOD (HSV-1)/3 x LOD (HSV-2) | 216 | 37.8 | 0.06 | 0.2 | 0.58 | 1.5 | 0.11 | 0.3 | 0.23 | 0.6 | 0.37 | 1.0 | 0.73 |
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-2 positive panel member "Below LOD (HSV-1/HSV-2)" was 56.5% (95% CI: 49.6% to 63.2%), whereas the positive percent agreement for all other positive panel members was 100.0% (95% CI: 98.3% to 100.0%). For the HSV-2 negative panel member, negative percent agreement was 100.0% (95% CI: 99.1% to 100.0%). The total SD and total CV (%) across all panel members were ≤ 0.92 and ≤ 2.3%, respectively.
{14}
# b. Precision:
The precision studies were conducted using three lots of reagents and three instrument for a total of 36 runs over 12 days. A six member study panel was prepared by spiking cultured HSV-1 (MacIntyre strain) and/or HSV-2 (G strain) virus into contrived sample matrix in transport media. The results from the precision study are presented in the tables below.
In-house precision study hit rate analysis
| 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 | Neg | Neg | 0 | 0 | 0 - 5% | 0 | 0 | 0 - 5% |
| P2 | < LOD | < LOD | 36 | 50 | 38 - 62% | 40 | 56 | 43 - 67% |
| P3 | ~ LOD | Neg | 72 | 100 | 95 - 100% | 0 | 0 | 0 - 5% |
| P4 | Neg | ~ 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% |
Variance components analysis for precision panel at $3 \times$ 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% |
{15}
Standard deviations and coefficients of variation (%) analysis for precision panel at 3 x LOD (Limit of Detection)
| 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% |
c. Linearity/assay reportable range: N/A
d. Traceability, Stability, Expected values (controls, calibrators, or methods):
## Assay Controls
Positive Control (PC): The Positive Control (PC) contains non-infectious DNA plasmids of both HSV-1 and HSV-2. The PC monitors the nucleic acid extraction, amplification, and detection steps in a given run of the test.
Negative Control (NC): The Negative Control (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.
Internal Control: The Internal Control is a lambda phage molecule that contains randomized sequences and targets for IC-specific primers and probe. The IC is added to all specimens and the positive and negative Controls during sample preparation on the cobas® x 480 instrument. The IC monitors nucleic acid extraction, amplification, and detection steps for a given specimen. The IC is also required for validation of the run controls.
## e. 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. The LoD of the cobas® HSV 1 and 2 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.
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HSV-1 MacIntyre and HSV-2 G strains were tested in the analytical sensitivity study. The final LoDs of the cobas® HSV 1 and 2 Test are presented in the table below.
Limit of Detection of cobas® HSV 1 and 2 Test
| Organism | Strain | ATCC ID | LoD (TCID_{50}/mL) |
| --- | --- | --- | --- |
| HSV-1 | MacIntyre | VR-539 | 0.479 |
| HSV-2 | G | VR-734 | 0.112 |
Analytical Inclusivity: In addition, 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 described in the Limit of Detection Section and was tested in 40 replicates near the LoD. All strains were detected by the assay, demonstrating that the cobas® HSV 1 and 2 Test can detect a broad range of both HSV-1 and HSV-2 strains.
f. Analytical Specificity/Cross Reactivity and Microbial Panel:
The analytical specificity/cross reactivity 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 consisted of bacteria, fungi viruses, human cells, and HSV-1 and HSV-2. Testing was performed with the organisms 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 three times of the LoD to assess the potential interference of the organisms cells on detection of HSV-1 and HSV-2 by the cobas® HSV 1 and 2 Test. All samples were prepared by diluting microorganisms or DNA into M4RT viral transport medium prior to testing for cross-reactivity.
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 concentration limitations.
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 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/mL; HHV-6A strain GS, HSV-1 and HSV-2 were quantified as viral particles, HIV-1 Strain IIIB and HBV were quantified as International Units (IU). HIV-2 Strain NIH-Z, Epstein-Barr Virus (HHV4), Varicella-Zoster Virus (HHV3) and
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HPV plasmids (HPV11, HPV16, 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.
No microorganisms tested positive for HSV-1 or HSV-2 using the cobas® HSV 1 and 2 Test when there was no HSV-1 and HSV-2 target present indicating no cross reactivity with these microorganisms. In addition, none of the organisms or a high concentration of human cells interfered with the detection of HSV-1 and HSV-2 targets. A high concentration of HSV-1 (1x10^6 vp/mL) did not produce false positive HSV-2 results and a high concentration of HSV-2 (1x10^6 vp/mL) did not produce false positive HSV-1 results.
Cross Reactivity & Microbial Interference Panel
| 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 | - |
* Two sources of Human Herpes Virus 8 were tested at 1.0 x 10^6 copies/mL (HHV8 viral DNA and HHV8 containing human cell line BCP-1).
** Chlamydia trachomatis serovar H was tested at 1.9 x 10^4 IFU/mL; Mycoplasma genitalium was tested at 1.0 x 10^5 CFU/mL; Treponema pallidum was tested at 9.0 x 10^4 copies/mL.
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# g. Interfering Studies
This study was performed to evaluate potential interference with the cobas® HSV 1 and 2 Test with twenty commonly used over the counter (OTC) products and antiviral medications, as well as whole blood, human serum albumin, urine, feces, and mucin. All OTC products were tested at or above (20 mg or 40 mg per swab for solids and 100% of swab capacity for liquids) 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 (maximum concentration of the drug as defined in the drug's labeling) in the collected specimen. HSV-1 and HSV-2 were spiked at ~ 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. The results are summarized in the table below.
Interfering Substances
| 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, | 20 mg, 40 mg |
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| Substance/Product Name | Composition | Testing Level/Swab |
| --- | --- | --- |
| | Potassium Hydroxide, Propylene Glycol, Purified Water, Stearyl Alcohol | |
| 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, Tocopherl Acetate, Retinyl Plamitate, 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 Monostearte, 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 |
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| Substance/Product Name | Composition | Testing Level/Swab |
| --- | --- | --- |
| Famciclovir | Famciclovir, Hydroxypropyl Cellulose, Hydroxypropyl Methylcellulose, Lactose, Magnesium Stearate, Polyethylene Glycols, Sodium Starch Glycolate, Titanium Dioxide | 0.016 mg |
| 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 |
# h. Competitive Inhibition
Competitive inhibition of the cobas® HSV 1 and 2 Test was evaluated to assess the potential interference in HSV-1/2 target detection when both HSV-1 and HSV-2 are present in a sample. The panels were constructed with HSV-1 at $\sim 3$ x LOD (Limit of Detection), and competing HSV-2 at $\sim 300$ 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 HSV-2 at approximately $3\mathrm{x}$ LOD concentration and one hundred fold higher concentration of HSV-2 did not affect the detection of HSV-1 at approximately $3\mathrm{x}$ LOD concentration.
# i. Carry-over/Cross Contamination
The "worst case use scenario" cross contamination rate for the cobas® HSV 1 and 2 Test was assessed by testing HSV-2 high positive and negative samples that were processed in a checkerboard configuration on cobas® 4800 system. High positive samples were prepared by adding HSV-2 plasmid (DNA) to MSwab medium to generate a Ct that exceeded the signal obtained in specimens from $95\%$ or more of infected patients in the intended use population.
Five runs were performed on each of the three cobas® 4800 systems. The first run on each system contained only the negative samples to confirm that the instrument was clean. The three subsequent runs had alternating positive and negative samples in checkerboard configurations to assess the cross contamination rate. The last run contained only the negative samples to assess the carry-over contamination rate.
In the nine checkerboard runs, 5 out of 422 HSV negative samples exhibited HSV-2 positive results, for an observed cross-contamination rate of $1.18\%$ . All results in the last 3 runs containing only the negative samples were negative, suggesting that there was no carry-over run-to-run contamination.
# j. Assay cut-off: Not applicable (N/A)
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22
2. Comparison studies:
a. Method comparison with predicate device:
The performance of the cobas® HSV 1/2 Test was compared to the composite reference method comprised of culture using the ELVIS® HSV ID/Typing Test System (Diagnostic Hybrids, Inc.) and PCR followed by bidirectional sequencing.
b. Matrix comparison: N/A
3. Clinical studies:
a. Clinical Sensitivity: N/A
b. Clinical Specificity: N/A
c. Other clinical supportive data (when a. and b. are not applicable):
The performance of the cobas® HSV 1 and 2 Test was compared to the composite reference method comprised of culture using the ELVIS® HSV ID/Typing Test System (Diagnostic Hybrids, Inc.) and PCR followed by bidirectional sequencing.
The clinical performance of the cobas® HSV 1 and 2 Test was also compared to the culture method alone using the FDA cleared ELVIS® HSV ID/Typing Test System.
## Clinical Performance
The clinical performance of the cobas® HSV 1 and 2 Test was established in a prospective, multi-site, investigation using the combined results of culture and PCR followed by bi-directional Sanger sequencing as the reference method. In addition, the performance of the cobas® HSV 1 and 2 Test was also compared to the culture method alone using the ELVIS® HSV ID and D³ Typing Test System.
A total of 408 prospective specimens (205 female and 203 male) were tested by the cobas® HSV 1 and 2 Test and compared to the combined results of culture (ELVIS® HSV ID/Typing Test System) and PCR followed by bidirectional sequencing. Two external anogenital swab specimens were collected from symptomatic eligible male and female subjects 17 years of age or older attending family planning, OB/GYN and sexually transmitted disease clinics at eight geographically diverse sites (seven across the United States and one in the United Kingdom). The first swab was used for (a) culture by the ELVIS® HSV ID and D³ Typing Test, (b) PCR followed by bidirectional Sanger sequencing for HSV-1 and HSV-2, and (c) discordant analysis by using an FDA cleared nucleic acid amplification test. The second swab was for the cobas® HSV 1 and 2 Test.
## Comparison with composite reference method (culture and Sanger sequencing)
A total of 408 prospective specimens collected from 205 female and 203 male subjects were evaluated in the study. There were 243 HSV positive subjects;
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84 HSV-1 (51 female, 33 male) and 167 HSV-2 (85 female, 82 male) positive subjects, with 8 (2%) subjects positive for both HSV-1 and HSV-2. The clinical performance of the cobas® HSV 1 and 2 Test compared to the composite reference method is presented in the table below.
Comparison of cobas® HSV 1 and 2 Test with the composite reference method
| | Composite Reference Method | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | HSV-1 | | | HSV-2 | | |
| | | Positive | Negative | Total | Positive | Negative | Total |
| cobas® HSV 1 and 2 Test | Positive | 78 | 4^{a} | 82 | 162 | 13^{c} | 175 |
| | Negative | 6^{b} | 320 | 326 | 5^{d} | 228 | 233 |
| | Total | 84 | 324 | 408 | 167 | 241 | 408 |
| HSV-1 | | | | HSV-2 | | | |
| Sensitivity: | 92.9% (78/84)
(95% CI = 85.3% - 96.7%) | | | Sensitivity: | 97.0% 162/167)
(95% CI = 93.2% - 98.7%) | | |
| Specificity: | 98.8% (320/324)
(95% CI = 96.9% - 99.5%) | | | Specificity: | 94.6% (228/241)
(95% CI = 91.0% - 96.8%) | | |
| PPV: | 95.1% (78/82)
(95% CI = 88.1% - 98.1%) | | | PPV: | 92.6% (162/175)
(95% CI = 87.7% - 95.6%) | | |
| NPV: | 98.2% (320/326)
(95% CI = 96.0% - 99.2%) | | | NPV: | 97.9% (228/233)
(95% CI = 95.1% - 99.1%) | | |
a Of the 4 specimens with HSV-1 false-positive cobas® HSV 1 and 2 Test results relative to the Reference Method, 2 were HSV-1 positive by a FDA-cleared nucleic acid amplification test.
b Of the 6 specimens with HSV-1 false-negative cobas® HSV 1 and 2 Test results relative to the Reference Method, all 6 were HSV-1 negative by a FDA-cleared nucleic acid amplification test.
c Of the 13 specimens with HSV-2 false-positive cobas® HSV 1 and 2 Test results relative to the Reference Method, 5 were HSV-2 positive by a FDA-cleared nucleic acid amplification test.
d Of the 5 specimens with HSV-2 false-negative cobas® HSV 1 and 2 Test results relative to the Reference Method, all 5 were HSV-2 negative by a FDA-cleared nucleic acid amplification test.
## Comparison with culture
The clinical performance of the cobas® HSV 1 and 2 Test was compared to the culture method alone using the ELVIS® HSV ID and D³ Typing Test system. The ELVIS® HSV ID and D³ Typing Test system used in this study is unable to detect patients co-infected with HSV-1 and HSV-2 and cannot detect HSV-1 if HSV-2 is detected first. Consequently, if a specimen was positive for HSV-2, it was removed from the calculation of the HSV-1 clinical performance. Only HSV-2 negative anogenital specimens were detected for HSV-1. Therefore, the number of samples used for the calculation of HSV-1 clinical performance equals the total number of specimens (408) minus the number of samples positive for HSV-2 by culture (129).
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The clinical performance of the cobas® HSV 1 and 2 Test compared to the culture reference method is presented in the table below.
Comparison of cobas® HSV 1 and 2 Test with culture
| | Culture Reference Method | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | HSV-1 | | | HSV-2 | | |
| | | Positive | Negative | Total | Positive | Negative | Total |
| cobas® HSV 1 and 2 Test | Positive | 67 | 13| 80 | 128 | 47^{}[] | 175 |
| | Negative | 0 | 199 | 199 | 1| 232 | 233 |
| | Total | 67 | 212 | 279 | 129 | 279 | 408 |
| HSV-1 | | | | HSV-2 | | | |
| Sensitivity: | 100.0% (67/67)
(95% CI = 94.6% - 100.0%) | | | Sensitivity: | 99.2% (128/129)
(95% CI = 95.7% - 99.9%) | | |
| Specificity: | 93.9% (199/212)
(95% CI = 89.8% - 96.4%) | | | Specificity: | 83.2% (232/279)
(95% CI = 78.3% - 87.1%) | | |
| PPV: | 83.8% (67/80)
(95% CI = 74.2% - 90.3%) | | | PPV: | 73.1% (128/175)
(95% CI = 66.1% - 79.2%) | | |
| NPV: | 100.0% (199/199)
(95% CI = 98.1% - 100.0%) | | | NPV: | 99.6% (232/233)
(95% CI = 97.6% - 99.9%) | | |
<a>^{}[]</a> The reference viral culture and typing method (ELVIS® HSV ID and D<sup>1</sup> Typing Test system) used in this study is unable to detect co-infected patients. Only HSV-2 negative specimens can be typed for HSV-1. Therefore, the number of samples used for the calculation of HSV-1 clinical performance equals the total number of evaluable specimens (408) minus the number of samples positive for HSV-2 by culture (129) for 279 evaluable specimens.
<a>^{}[]</a> Of the 13 specimens with HSV 1 false-positive cobas® HSV 1 and 2 Test results relative to the culture and typing, 6 were HSV-1 positive by a FDA-cleared nucleic acid amplification test and 4 of which were also HSV-1 positive by Sanger sequencing; 5 additional samples were HSV-1 positive by Sanger sequencing alone.
<a>^{}[]</a> Of the 47 specimens with HSV 2 false-positive cobas® HSV 1 and 2 Test results relative to the culture and typing, 32 were HSV-2 positive by a FDA-cleared nucleic acid amplification test.
<a>^{}[]</a> The one specimen with a HSV 2 false-negative cobas® HSV 1 and 2 Test result relative to the culture and typing was HSV-2 negative by a FDA-cleared nucleic acid amplification test.
Note: CI = (Score) confidence interval, PPV = positive predictive value, NPV = negative predictive value.
4. Clinical cut-off: N/A
5. Expected values/Reference range:
**Prevalence**
The prevalence of HSV-1 and HSV-2 observed during the multi-center clinical trial was calculated for the cobas® HSV 1 and 2 Test. The prevalence rates for HSV-1 and HSV-2 were individually established as 20.6% (84/408) and 40.9% (167/408) for anogenital samples. The gender and age distribution from anogenital specimens and the expected prevalence values for the cobas® HSV 1 and 2 Test from anogenital specimens is shown
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by age for males and females in the tables below.
## Gender and Age Distribution for the cobas® HSV 1 and 2 Test from anogenital specimens (N = 408 evaluable results)
| Age | Gender | | Total (N) |
| --- | --- | --- | --- |
| | Male (n) | Female (n) | |
| 17 | 0 | 2 | 2 |
| 18 to <21 | 15 | 41 | 56 |
| ≥21 | 188 | 162 | 350 |
| Total (N) | 203 | 205 | 408 |
## Expected Prevalence Values for the cobas® HSV 1 and 2 Test for anogenital specimens from males by age (n = 203 evaluable results)
| Age | Total (N) | HSV-1 | | HSV-2 | |
| --- | --- | --- | --- | --- | --- |
| | | Positive (n) | Prevalence (%) | Positive (n) | Prevalence (%) |
| 17 | 0 | 0 | 0 | 0 | 0 |
| 18 to <21 | 15 | 3 | 20.0 | 5 | 33.3 |
| ≥21 | 188 | 30 | 16.0 | 77 | 41.0 |
| Total | 203 | 33 | 16.3 | 82 | 40.4 |
## Expected Prevalence Values for the cobas® HSV 1 and 2 Test for anogenital specimens from females by age (n = 205 evaluable results)
| Age | Total (N) | HSV-1 | | HSV-2 | |
| --- | --- | --- | --- | --- | --- |
| | | Positive (n) | Prevalence (%) | Positive (n) | Prevalence (%) |
| 17 | 2 | 0 | 0 | 1 | 50.0 |
| 18 to <21 | 41 | 17 | 41.5 | 13 | 31.7 |
| ≥21 | 162 | 34 | 21.0 | 71 | 43.8 |
| Total | 205 | 51 | 24.9 | 85 | 41.5 |
## Positive and Negative Predictive Value
Hypothetical positive and negative predictive values (PPV & NPV) for the cobas® HSV 1 and 2 Test are shown in the table below. These calculations are based on hypothetical prevalence and overall sensitivity and specificity for anogenital swab specimens as determined in the clinical trial.
For HSV-1, the calculations are based upon an overall sensitivity and specificity of 92.9% and 98.8%, respectively.
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For HSV-2, these calculations are based upon an overall sensitivity and specificity of 97.0% and 94.6%, respectively, for anogenital swabs.
Hypothetical Positive and Negative Predictive Values (PPV & NPV) for the cobas® HSV 1 and 2 Test from anogenital specimens
| Hypothetical Prevalence (%) | HSV-1 | | HSV-2 | |
| --- | --- | --- | --- | --- |
| | PPV^{a} (%) | NPV^{b} (%) | PPV^{a} (%) | NPV^{b} (%) |
| 5 | 79.8 | 99.6 | 48.6 | 99.8 |
| 10 | 89.3 | 99.2 | 66.6 | 99.6 |
| 15 | 93.0 | 98.7 | 76.0 | 99.4 |
| 20 | 95.0 | 98.2 | 81.8 | 99.2 |
| 25 | 96.2 | 97.6 | 85.7 | 99.0 |
| 30 | 97.0 | 97.0 | 88.5 | 98.7 |
| 40 | 98.0 | 95.4 | 92.3 | 97.9 |
a PPV = (Sensitivity x Prevalence) / (Sensitivity x Prevalence + [1 - Specificity] x [1 - Prevalence]).
b NPV = (Specificity x [1 - Prevalence]) / ([1 - Sensitivity] x Prevalence + Specificity x [1 - Prevalence]).
N. Instrument Name
cobas® 4800 System
O. System Descriptions:
1. Modes of Operation:
Does the applicant’s device contain the ability to transmit data to a computer, webserver, or mobile device?
Yes ☐ or No ☑
Does the applicant’s device transmit data to a computer, webserver, or mobile device using wireless transmission?
Yes ☐ or No ☐
2. Software:
FDA has reviewed applicant’s Hazard Analysis and software development processes for this line of product types:
Yes ☐ or No ☑
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3. Specimen Identification:
Specimens are identified using barcodes on specimen vials.
4. Specimen Sampling and Handling:
Specimens are placed on the cobas x 480 instrument as open tubes and specimen processing is fully automated. After completion of specimen processing, the user transfers the plate carrier to the cobas z 480 instrument for automated amplification and detection. Specimens can be processed directly from primary collection vials or as aliquots of the specimen in secondary vials. See section I for more information on specimen handling.
5. Calibration:
No calibration is required by the user. Roche technicians perform calibration periodically as required.
6. Quality Control:
See section M.1.d for information on internal and external controls.
P. Proposed Labeling:
The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.
Q. Conclusion:
The submitted information in this premarket notification is complete and supports a substantial equivalence decision.
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