Simplexa Congenital CMV Direct and Simplexa Congenital CMV Positive Control Pack

K202755 · Diasorin Molecular, LLC · QDZ · Nov 5, 2022 · Microbiology

Device Facts

• Record ID: K202755
• Device Name: Simplexa Congenital CMV Direct and Simplexa Congenital CMV Positive Control Pack
• Applicant: Diasorin Molecular, LLC
• Product Code: QDZ · Microbiology
• Decision Date: Nov 5, 2022
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.3181
• Device Class: Class 2
• Attributes: Pediatric

Indications for Use

The DiaSorin Molecular Simplexa™ Congenital CMV Direct is a real-time PCR assay intended for use on the LIAISON® MDX instrument for the in vitro qualitative detection of cytomegalovirus (CMV) from saliva swabs and urine from infants less than 21 days of age. Positive results from saliva are presumptive and should be confirmed with urine. The results of the Simplexa™ Congenital CMV Direct assay should be used in conjunction with the results of other clinical findings as an aid in the diagnosis of congenital CMV infection. This test has not been cleared for screening of blood products for the presence of CMV or for use with samples other than urine and saliva swabs. DiaSorin Molecular's Simplexa™ Congental CMV Positive Control Pack is intended to be used as a control with the Simplexa Congenital CMV Direct kit for use on the LIAISON MDX instrument. This control is not intended for use with other assays or systems.

Device Story

Real-time PCR assay for direct amplification/detection of CMV DNA from saliva swabs or urine; no nucleic acid extraction required. Uses bi-functional fluorescent probe-primers targeting CMV UL83 gene; internal control monitors for PCR failure/inhibition. Performed on LIAISON MDX instrument. Results aid clinicians in diagnosing congenital CMV infection in neonates. Positive saliva results require urine confirmation. Used in clinical laboratory settings by trained personnel. Output is qualitative detection of CMV DNA.

Clinical Evidence

Clinical performance evaluated via retrospective (n=346) and prospective (n=3,515) studies using a Composite Reference Method (CRM) of two validated PCR/bi-directional sequencing assays. Retrospective saliva PPA 100%, NPA 100%; urine PPA 100%, NPA 98.4%. Prospective saliva PPA 94.1%, NPA 99.9%; urine PPA 95.3%, NPA 100%.

Technological Characteristics

Real-time PCR assay; targets CMV UL83 gene; uses bi-functional fluorescent probe-primers. Direct amplification (no extraction). Instrument: LIAISON MDX. Connectivity: Standalone. Software: LIAISON MDX Studio Software. Sterilization: N/A (reagents).

Indications for Use

Indicated for infants < 21 days of age for the qualitative detection of CMV DNA in saliva swabs and urine to aid in the diagnosis of congenital CMV infection.

Regulatory Classification

Identification

A cytomegalovirus nucleic acid detection device for congenital cytomegalovirus infection is an in vitro diagnostic device intended for the qualitative detection of cytomegalovirus DNA in clinical samples from newborn babies to aid in the diagnosis of congenital cytomegalovirus infection. Negative results do not preclude infection and should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. Positive results should be interpreted with consideration of other clinical information and laboratory findings and should not be used as the sole basis for treatment or other patient management decisions.

Special Controls

The special controls for this device are: (1) The 21 CFR 809.10 labeling must include: (i) An intended use with a detailed description of what the device detects, the type of results provided to the user, the clinical indications appropriate for test use, and the specific population(s) to be tested. (ii) A detailed device description, including all device components, instrument requirements, ancillary reagents required but not provided, and an explanation of the methodology, including all pre-analytical methods for specimen processing. (iii) Performance characteristics from analytical and clinical studies required under paragraphs (b)(2)(ii) and (b)(2)(iii) of this section. (iv) A detailed explanation of the interpretation of results and criteria for validity of results. (v) A limiting statement that device results are not intended to be used as the sole basis for diagnosis, treatment, or other patient management decisions. (vi) As applicable, a limiting statement and specific sample collection recommendations to indicate that breast milk can result in false positive results for saliva samples if samples are collected less than one hour after breastfeeding. Sample collection a minimum of one hour from breastfeeding must be recommended. (vii) Detailed instructions for use that minimize the risk of generating a false result. (2) Design verification and validation must include: Detailed device description documentation, including but not limited to, (i) methodology from obtaining sample to result, design of primer/probe sequences, rationale for sequence selection, and computational path from collected raw data to reported result (e.g., how collected raw signals are converted into a reported result). Detailed documentation of analytical studies including but not limited to, (ii) characterization of the cut-off, analytical sensitivity (limit of detection), inclusivity, reproducibility, interference, cross reactivity, instrument and method carryover/cross contamination, sample stability and handling. (iii) Detailed documentation from a clinical study documenting sensitivity and specificity of the device; if the number of positive samples in the clinical study is insufficient to properly estimate device sensitivity, additional pre-selected positive samples must be evaluated to supplement the study. Clinical study subjects must be consistent with the intended use population (i.e., infants younger than 21 days of age), and device results must be compared to FDA-accepted comparator methods. Documentation from the clinical study must include the clinical study protocol, the clinical study report, testing results, and results of all statistical analyses. (iv) Detailed documentation for device software, including, but not limited to, software applications and hardware-based devices that incorporate software.

*Classification.* Class II (special controls). The special controls for this device are:(1) The labeling required under § 809.10(b) of this chapter must include: (i) An intended use with a detailed description of what the device detects, the type of results provided to the user, the clinical indications appropriate for test use, and the specific population(s) to be tested. (ii) A detailed device description, including all device components, instrument requirements, ancillary reagents required but not provided, and an explanation of the methodology, including all pre-analytical methods for specimen processing. (iii) Performance characteristics from analytical and clinical studies required under paragraphs (b)(2)(ii) and (iii) of this section. (iv) A detailed explanation of the interpretation of results and criteria for validity of results. (v) A limiting statement that device results are not intended to be used as the sole basis for diagnosis, treatment, or other patient management decisions. (vi) As applicable, a limiting statement and specific sample collection recommendations to indicate that breast milk can result in false positive results for saliva samples if samples are collected less than 1 hour after breastfeeding. Sample collection a minimum of 1 hour from breastfeeding must be recommended. (vii) Detailed instructions for use that minimize the risk of generating a false result. (2) Design verification and validation must include: (i) Detailed device description documentation, including methodology from obtaining sample to result, design of primer/probe sequences, rationale for sequence selection, and computational path from collected raw data to reported result ( *e.g.,* how collected raw signals are converted into a reported result).(ii) Detailed documentation of analytical studies including characterization of the cutoff, analytical sensitivity (limit of detection), inclusivity, reproducibility, interference, cross reactivity, instrument and method carryover/cross contamination, and sample stability and handling. (iii) Detailed documentation from a clinical study documenting sensitivity and specificity of the device; if the number of positive samples in the clinical study is insufficient to properly estimate device sensitivity, additional pre-selected positive samples must be evaluated to supplement the study. Clinical study subjects must be consistent with the intended use population ( *i.e.,* infants younger than 21 days of age), and device results must be compared to FDA-accepted comparator methods. Documentation from the clinical study must include the clinical study protocol, the clinical study report, testing results, and results of all statistical analyses.(iv) Detailed documentation for device software, including software applications and hardware-based devices that incorporate software.

Predicate Devices

• Alethia® CMV Assay Test System (DEN180040)

Submission Summary (Full Text)

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FDA U.S. FOOD &amp; DRUG ADMINISTRATION

# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

## I Background Information:

A 510(k) Number

K202755

B Applicant

DiaSorin Molecular LLC

C Proprietary and Established Names

Simplexa Congenital CMV Direct and Simplexa Congenital CMV Positive Control Pack

D Regulatory Information

| Product Code(s) | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| QDZ | Class II | 21 CFR 866.3181 - Cytomegalovirus Nucleic Acid Detection Device For Congenital Cytomegalovirus Infection | MI - Microbiology |

## II Submission/Device Overview:

A Purpose for Submission:

The sole purpose of the application is to support conditions of use of the Simplexa Congenital CMV Direct for a neonate population less than 21 days old.

B Measurand:

CMV DNA

C Type of Test:

Polymerase chain reaction (PCR)

Food and Drug Administration

10903 New Hampshire Avenue

Silver Spring, MD 20993-0002

www.fda.gov

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III Intended Use/Indications for Use:

A Intended Use(s):

The DiaSorin Molecular Simplexa Congenital CMV Direct is a real-time PCR assay intended for use on the LIAISON MDX instrument for the *in vitro* qualitative detection of cytomegalovirus (CMV) from saliva swabs and urine from infants less than 21 days of age. Positive results from saliva are presumptive and should be confirmed with urine. The results of the Simplexa Congenital CMV Direct assay should be used in conjunction with the results of other clinical findings as an aid in the diagnosis of congenital CMV infection. This test has not been cleared for screening of blood or blood products for the presence of CMV or for use with samples other than urine and saliva swabs.

Simplexa Congenital CMV Positive Control Pack Catalog Number MOL2260 DiaSorin Molecular’s Simplexa Congenital CMV Positive Control Pack is intended to be used as a control with the Simplexa Congenital CMV Direct kit for use on the LIAISON MDX instrument. This control is not intended for use with other assays or systems.

B Indication(s) for Use:
NA

C Special Conditions for Use Statement(s):
Rx

D Special Instrument Requirements:
LIAISON MDX

IV Device/System Characteristics:

A Device Description:

The Simplexa Congenital CMV Direct assay is a real-time PCR system that enables the direct amplification and detection of CMV DNA from either saliva swab or urine specimens without nucleic acid extraction. The system consists of the Simplexa Congenital CMV Direct Reaction Mix, the LIAISON MDX (with LIAISON® MDX Studio Software), the Direct Amplification Disc (DAD) and associated accessories.

In the Simplexa Congenital CMV Direct assay, bi-functional fluorescent probe-primers are used together with corresponding reverse primers to amplify CMV DNA. A well-conserved region of the CMV UL83 gene is targeted to identify CMV DNA. An internal control is used to detect PCR failure and/or inhibition.

B Principle of Operation:

Real-time PCR

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C Instrument Description Information:

1. Instrument Name:
LIAISON MDX

2. Specimen Identification:
NA

3. Specimen Sampling and Handling:
Acceptable specimen type is/are either urine collected from urine bags/containers/catheters or saliva flocked swab specimens transported in BD UVT (1mL or 3mL), Copan UTM (1mL or 3mL), Remel M4RT, M4 and M6 transport media.

Follow the manufacturers' package inserts for collection media and acceptable flocked swab types. Do not use calcium alginate or cotton swabs, as they may contain substances that inhibit PCR testing.

Urine specimens should be processed within 2 hours post collection if stored at 18°C to 25°C, or within 8 hours post collection if transported on ice and stored at 2 to 8°C. If there is a greater than 8 hours delay before processing the specimen, store the specimen for up to 3 days at -20 ± 10 °C. If there is a greater than 3 days delay before testing, store the specimen at ≤ -70 °C.

Saliva swab specimens should be processed within 48 hours post collection if stored at 18°C to 25°C, or within 7 days post collection if transported on ice and stored at 2 to 8°C. If there is a greater than 7 days delay before processing the specimen, store the specimen for up to 14 days at -20 ±10 °C. If there is a greater than 14 days delay before testing, store specimen at ≤ C.

4. Calibration:
NA

5. Quality Control:
Simplexa Congenital CMV Positive Control Pack (MOL2260) may be used as an external control for Quality Control (QC) testing, training or proficiency testing. Each laboratory should establish its own QC ranges and frequency of QC testing based on applicable local laws, regulations and standard good laboratory practice. Refer to the Simplexa Congenital CMV Positive Control Pack (IFUC.US.MOL2260) for instructions on testing the Positive Control.

V Substantial Equivalence Information:

A Predicate Device Name(s):
Alethia CMV DNA Amplification Assay, Alethia CMV External Control Kit

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B Predicate 510(k) Number(s):

DEN180040

C Comparison with Predicate(s):

| Device & Predicate Device(s): | K202755 | DEN180040 |
| --- | --- | --- |
| Device Trade Name | Simplexa Congenital CMV Direct and Simplexa Congenital CMV Positive Control Pack | Predicate Device: Alethia CMV Assay Test System (DEN180040) |
| General Device Characteristic Similarities | | |
| Organism Detected | Cytomegalovirus | same |
| Measurand | CMV DNA | same |
| | | |
| General Device Characteristic Differences | | |
| Intended Use | The DiaSorin Molecular Simplexa Congenital CMV Direct is a real-time PCR assay intended for use on the LIAISON MDX instrument for the in vitro qualitative detection of cytomegalovirus (CMV) from saliva swabs and urine from infants less than 21 days of age. Positive results from saliva are presumptive and should be confirmed with urine. The results of the Simplexa Congenital CMV Direct assay should be used in conjunction with the results of other clinical findings as an aid in the diagnosis of congenital CMV infection. This test has not been cleared for screening of blood | The Alethia CMV Assay Test System includes separately provided test kits for the Alethia CMV DNA Amplification Assay and the Alethia CMV External Control Reagents. The Alethia CMV DNA Amplification Assay, performed on the Alethia instrument, is a qualitative, in vitro diagnostic test system for the direct detection of Cytomegalovirus (CMV) DNA in saliva samples from neonates younger than 21 days of age. The test is used as an aid in the diagnosis of congenital CMV infection. The results of this test should be used in conjunction with the |

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| | or blood products for the presence of CMV or for use with samples other than urine and saliva swabs.

Simplexa Congenital CMV Positive Control Pack Catalog Number MOL2260 DiaSorin Molecular’s Simplexa Congenital CMV Positive Control Pack is intended to be used as a control with the Simplexa Congenital CMV Direct kit for use on the LIAISON MDX instrument. This control is not intended for use with other assays or systems. | results of other clinical findings.

Flocked swabs should be used to collect saliva from neonates. The swab can be collected dry, without viral transport media (VTM), or placed in no more than 1 mL VTM. The Alethia CMV External Control Reagents are used as part of a routine quality control program to aid the user in detection of unexpected conditions that may lead to test errors. The external controls are intended for use with the Alethia CMV DNA Amplification Assay; the controls are not intended for use with other assays or systems. |
| --- | --- | --- |
| Instrumentation | LIAISON MDX | Alethia Instrument; Meridian Bioscience, Inc. |
| Sample Types/Media Type | Saliva swab in BD Universal Viral Transport (UVT), Copan UTM (1mL or 3mL), Remel M4RT, M4, and M6 transport and urine from infants less than 21 days old. | Dry flocked saliva swab or saliva swab in 1 mL VTM from infants < 21 days old. |

VI Standards/Guidance Documents Referenced:

NA

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VII Performance Characteristics (if/when applicable):

A Analytical Performance:

1. Precision/Reproducibility:

REPRODUCIBILITY

Reproducibility for the Simplexa Congenital CMV Direct assay was evaluated. Three (3) investigative sites assessed the device's inter-site, inter-day and inter/intra-assay reproducibility. Each of the sites tested the Simplexa Congenital CMV Direct Positive Control, No Template Control (NTC), a negative urine sample, a negative saliva swab in UTM sample and eight (8) contrived samples spiked into a negative matrix of either saliva swabs in UTM or urine. The eight (8) contrived samples consisted of a low positive (LP) contrived at approximately 3X the limit of detection (LOD) and a medium positive (MP) contrived at approximately 10X LOD for each of the following: CMV strain AD169 in saliva swabs in UTM, CMV strain AD169 in urine, CMV strain Towne in saliva swabs in UTM, and CMV strain Towne in urine. Each contrived sample was prepared by spiking a specific concentration of the strain into CMV negative urine or a CMV negative saliva swab in UTM. The samples were tested in quadruplicate on nine (9) different days. Each site had three (3) operators who each assayed the entire sample panel and Positive Control twice per day, for a total of two (2) sets of data per day on one (1) LIAISON® MDX instrument, per site. The combined results for all sites are presented in Table 5. The results show the reproducibility of the Simplexa Congenital CMV Direct % CV ranged between 0.4-1.6%, Table 1.

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FDA

U.S. FOOD &amp; DRUG

ADMINISTRATION

Table 1. Simplexa Congenital CMV Direct Reproducibility

| Sample CMV Strain and Matrix | Site 1 | | Site 2 | | Site 3 | | All Sites | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | Agreement with expected results | Avg. Ct ± SD (%CV) | Agreement with expected results | Avg. Ct ± SD (%CV) | Agreement with expected results | Avg. Ct ± SD (%CV) | Agreement with expected results | Avg. Ct ± SD (%CV) |
| Saliva Towne_LP | 100.0% (36/36) | 34.3 ± 0.32 (0.9%) | 100.0% (36/36) | 34.5 ± 0.26 (0.7%) | 100.0% (36/36) | 34.3 ± 0.32 (0.9%) | 100.0% (108/108) | 34.4 ± 0.32 (0.9%) |
| Saliva Towne_MP | 100.0% (36/36) | 32.2 ± 0.25 (0.8%) | 100.0% (36/36) | 32.5 ± 0.21 (0.6%) | 100.0% (36/36) | 32.2 ± 0.22 (0.7%) | 100.0% (108/108) | 32.3 ± 0.25 (0.8%) |
| Saliva AD-169_LP | 100.0% (36/36) | 33.4 ± 0.34 (1.0%) | 100.0% (36/36) | 33.8 ± 0.23 (0.7%) | 100.0% (36/36) | 33.5 ± 0.25 (0.8%) | 100.0% (108/108) | 33.6 ± 0.32 (1.0%) |
| Saliva AD-169_MP | 100.0% (36/36) | 32.2 ± 0.22 (0.7%) | 100.0% (36/36) | 32.3 ± 0.27 (0.8%) | 100.0% (36/36) | 32.1 ± 0.15 (0.5%) | 100.0% (108/108) | 32.2 ± 0.23 (0.7%) |
| Urine Towne_LP | 100.0% (36/36) | 33.9 ± 0.51 (1.5%) | 100.0% (36/36) | 34.0 ± 0.48 (1.4%) | 100.0% (36/36) | 33.9 ± 0.45 (1.3%) | 100.0% (108/108) | 34.0 ± 0.48 (1.4%) |
| Urine Towne_MP | 100.0% (36/36) | 32.1 ± 0.26 (0.8%) | 100.0% (36/36) | 32.3 ± 0.26 (0.8%) | 100.0% (36/36) | 32.1 ± 0.21 (0.7%) | 100.0% (108/108) | 32.2 ± 0.26 (0.8%) |
| Urine AD-169_LP | 100.0% (36/36) | 35.9 ± 0.57 (1.6%) | 100.0% (36/36) | 36.1 ± 0.59 (1.6%) | 97.2% (35/36) | 35.9 ± 0.46 (1.3%) | 99.1% (107/108) | 36.0 ± 0.54 (1.5%) |
| Urine AD-169_MP | 100.0% (36/36) | 34.0 ± 0.42 (1.2%) | 100.0% (36/36) | 34.3 ± 0.37 (1.1%) | 100.0% (36/36) | 34.0 ± 0.33 (1.0%) | 100.0% (108/108) | 34.1 ± 0.40 (1.2%) |
| Saliva_Negative* | 100.0% (36/36) | 0.0 ± 0.00 (N/A%) | 100.0% (36/36) | 0.0 ± 0.00 (N/A%) | 100.0% (36/36) | 0.0 ± 0.00 (N/A%) | 100.0% (108/108) | 0.0 ± 0.00 (N/A%) |
| Urine_Negative* | 100.0% (36/36) | 0.0 ± 0.00 (N/A%) | 100.0% (36/36) | 0.0 ± 0.00 (N/A%) | 100.0% (36/36) | 0.0 ± 0.00 (N/A%) | 100.0% (108/108) | 0.0 ± 0.00 (N/A%) |
| NTC (UTM) | 100.0% (36/36) | 0.0 ± 0.00 (N/A%) | 100.0% (36/36) | 0.0 ± 0.00 (N/A%) | 100.0% (36/36) | 0.0 ± 0.00 (N/A%) | 100.0% (108/108) | 0.0 ± 0.00 (N/A%) |
| PC as is | 100.0% (36/36) | 29.7 ± 0.14 (0.5%) | 100.0% (36/36) | 29.9 ± 0.13 (0.4%) | 100.0% (36/36) | 29.6 ± 0.22 (0.7%) | 100.0% (108/108) | 29.7 ± 0.20 (0.7%) |

Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
www.fda.gov

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FDA U.S. FOOD &amp; DRUG ADMINISTRATION

2. Linearity:

NA

3. Analytical Specificity/Interference:

Cross-Reactivity (Analytical Specificity)

The Simplexa Congenital CMV Direct assay's analytical specificity was evaluated by testing the ability of the assay to exclusively identify CMV virus with no cross-reactivity to organisms that are closely related, cause similar clinical symptoms or may be present in saliva swabs and urine. Forty-one (41) potential cross-reactants were spiked into negative saliva swab and thirteen (13) potential cross reactants were spiked into negative urine. The samples were assayed in triplicate. No cross-reactivity was observed. The results are presented in Tables 2a and 2b.

Table 2a. Simplexa Congenital CMV Direct Cross-Reactivity (Analytical Specificity) - Saliva Swab

| No. | Organism | Tested Concentration | % Agreement (# Expected Results/# Tested) |
| --- | --- | --- | --- |
| 1 | Acinetobacter baumannii | 1x10^{6} CFU/mL | 100.0% (3/3) |
| 2 | Actinomyces odontolyticus | 1x10^{6} CFU/mL | 100.0% (3/3) |
| 3 | Bordetella pertussis | 1x10^{6} CFU/mL | 100.0% (3/3) |
| 4 | Coronavirus 229E | 1x10^{5} TCID50/mL | 100.0% (3/3) |
| 5 | Coxsackievirus A9 | 1x10^{5} TCID50/mL | 100.0% (3/3) |
| 6 | Epstein-Barr Virus | 1x10^{5} IU/mL | 100.0% (3/3) |
| 7 | Enterovirus 71* | 1x10^{4} TCID50/mL | 100.0% (3/3) |
| 8 | FLU A/ Michigan/45/2 015 | 1x10^{5} Cps/mL | 100.0% (3/3) |
| 9 | FLU B/ Phuket/3073/2 013 | 1x10^{5} Cps/mL | 100.0% (3/3) |
| 10 | Fusobacterium nucleatum | 1x10^{6} CFU/mL | 100.0% (3/3) |
| 11 | Adenovirus (C1) | 1x10^{5} Cps/mL | 100.0% (3/3) |
| 12 | Haemophilus influenza | 1x10^{6} CFU/mL | 100.0% (3/3) |
| 13 | Haemophilus parainfluenzae | 1x10^{6} CFU/mL | 100.0% (3/3) |
| 14 | Herpes Simplex Virus 1 | 1x10^{5} IU/mL | 100.0% (3/3) |
| 15 | Human herpesvirus 6A | 1x10^{5} Cps/mL | 100.0% (3/3) |

Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
www.fda.gov

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| 16 | Human herpesvirus 6B | 1x10^{5} Cps/mL | 100.0% (3/3) |
| --- | --- | --- | --- |
| 17 | Human herpesvirus 7 | 1x10^{5} Cps/mL | 100.0% (3/3) |
| 18 | Human herpesvirus 8 | 1x10^{5} Cps/mL | 100.0% (3/3) |
| 19 | Human Genomic DNA | 1x10^{6} Cps/mL | 100.0% (3/3) |
| 20 | Human metapneumovirus | 1x10^{5} TCID50/mL | 100.0% (3/3) |
| 21 | Klebsiella oxytoca | 1x10^{6} CFU/mL | 100.0% (3/3) |
| 22 | Klebsiella pneumoniae | 1x10^{6} CFU/mL | 100.0% (3/3) |
| 23 | Moraxella catarrhalis | 1x10^{6} CFU/mL | 100.0% (3/3) |
| 24 | Mycoplasma pneumoniae | 1x10^{6} CCU/mL | 100.0% (3/3) |
| 25 | Parainfluenza virus 1 | 1x10^{5} U/mL | 100.0% (3/3) |
| 26 | Parainfluenza virus 2 | 1x10^{5} U/mL | 100.0% (3/3) |
| 27 | Parainfluenza virus 3 | 1x10^{5} U/mL | 100.0% (3/3) |
| 28 | Porphyromonas gingivalis | 1x10^{6} CFU/mL | 100.0% (3/3) |
| 29 | Pseudomonas aeruginosa | 1x10^{6} CFU/mL | 100.0% (3/3) |
| 30 | Respiratory syncytial virus A | 1x10^{5} IU/mL | 100.0% (3/3) |
| 31 | Respiratory syncytial virus B | 1x10^{5} TCID50/mL | 100.0% (3/3) |
| 32 | Rhinovirus | 1x10^{5} U/mL | 100.0% (3/3) |
| 33 | Staphylococcus aureus | 1x10^{6} CFU/mL | 100.0% (3/3) |
| 34 | Staphylococcus epidermidis | 1x10^{6} CFU/mL | 100.0% (3/3) |
| 35 | Streptococcus anginosus | 1x10^{6} CFU/mL | 100.0% (3/3) |
| 36 | Streptococcus oralis | 1x10^{6} CFU/mL | 100.0% (3/3) |
| 37 | Streptococcus mitis | 1x10^{6} CFU/mL | 100.0% (3/3) |
| 38 | Streptococcus pneumoniae | 1x10^{6} CFU/mL | 100.0% (3/3) |
| 39 | Streptococcus salivarius | 1x10^{6} CFU/mL | 100.0% (3/3) |
| 40 | Streptococcus sanguinis | 1x10^{6} CFU/mL | 100.0% (3/3) |
| 41 | Varicella Zoster Virus | 1x10^{5} Cps/mL | 100.0% (3/3) |

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Table 2b. Simplexa Congenital CMV Direct Cross-Reactivity (Analytical Specificity) - Urine

| No. | Organism | Tested Concentration | % Agreement (# Expected Results/ # Tested) |
| --- | --- | --- | --- |
| 1 | Candida albicans | 1x106 CFU/mL | 100.0% (3/3) |
| 2 | Enterobacter aerogenes | 1x106 CFU/mL | 100.0% (3/3) |
| 3 | Enterobacter cloacae | 1x106 CFU/mL | 100.0% (3/3) |
| 4 | Enterococcus faecium | 1x106 CFU/mL | 100.0% (3/3) |
| 5 | Enterococcus faecalis | 1x106 CFU/mL | 100.0% (3/3) |
| 6 | Escherichia coli | 1x106 CFU/mL | 100.0% (3/3) |
| 7 | Herpes Simplex Virus 2 | 1x105 cps/mL | 100.0% (3/3) |
| 8 | Lactobacillus acidophilus | 1x106 CFU/mL | 100.0% (3/3) |
| 9 | Morganella morganii | 1x106 CFU/mL | 100.0% (3/3) |
| 10 | Proteus mirabilis | 1x106 CFU/mL | 100.0% (3/3) |
| 11 | Proteus vulgaris | 1x106 CFU/mL | 100.0% (3/3) |
| 12 | Streptococcus agalactiae (GBS) | 1x106 CFU/mL | 100.0% (3/3) |
| 13 | Enterovirus 71* | 1x10^{4} TCID50/mL | 100.0% (3/3) |

# INTERFERENCE

The performance of the Simplexa Congenital CMV Direct assay was evaluated with potentially interfering substances that may be present in saliva swabs and urine samples at the concentrations indicated in the table below. A total of seventeen (17) potentially interfering substances were tested for saliva swabs and seven (7) potentially interfering substances were tested for urine in a low positive CMV sample at approximately three times the limit of detection (3X LoD) in saliva swab and urine matrices and assayed in triplicate. No interference was observed. The results are presented in Tables 3a and 3b.

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Table 3a. Simplexa Congenital CMV Direct Interference for saliva swab

| No. | Potential Interferent | CMV Strain | Tested Concentration | % Agreement* (# Expected Results/# Tested) |
| --- | --- | --- | --- | --- |
| 1 | Acetylsalicylic acid | AD169 | 0.65 mg/mL | 100.0% (3/3) |
| | | Towne | 0.65 mg/mL | 100.0% (3/3) |
| 2 | Breast milk | AD169 | 10% (v/v) | 100.0% (3/3) |
| | | Towne | 10% (v/v) | 100.0% (3/3) |
| 3 | Caffeine | AD169 | 0.06 mg/mL | 100.0% (3/3) |
| | | Towne | 0.06 mg/mL | 100.0% (3/3) |
| 4 | Casein | AD169 | 10 mg/mL | 100.0% (3/3) |
| | | Towne | 10 mg/mL | 100.0% (3/3) |
| 5 | Enfamil Poly-vi-sol with Iron | AD169 | 1.5 mg/mL | 100.0% (3/3) |
| | | Towne | 1.5 mg/mL | 100.0% (3/3) |
| 6 | Enfamil™ formula neuro pro® | AD169 | 10% (v/v) | 100.0% (3/3) |
| | | Towne | 10% (v/v) | 100.0% (3/3) |
| 7 | Enfamil™ Tri-Vi-Sol® | AD169 | 8% (v/v) | 100.0% (3/3) |
| | | Towne | 8% (v/v) | 100.0% (3/3) |
| 8 | Gaviscon® (Sodium Alginate) | AD169 | 1.2 mg/mL | 100.0% (3/3) |
| | | Towne | 1.2 mg/mL | 100.0% (3/3) |
| 9 | Infants' Pain & Fever (Acetaminophen) | AD169 | 0.4 mg/mL | 100.0% (3/3) |
| | | Towne | 0.2 mg/mL | 100.0% (3/3) |
| 10 | Infants' Mylicon® Gas Relief (Simethicone) | AD169 | 6.7 mg/mL | 100.0% (3/3) |
| | | Towne | 6.7 mg/mL | 100.0% (3/3) |
| 11 | Little Remedies Saline Drops | AD169 | 10% (v/v) | 100.0% (3/3) |
| | | Towne | 10% (v/v) | 100.0% (3/3) |
| 12 | Motrin Infant Drops (Infants' Ibuprofen) | AD169 | 0.5 mg/mL | 100.0% (3/3) |
| | | Towne | 0.5 mg/mL | 100.0% (3/3) |
| 13 | Mucin | AD169 | 25 mg/mL | 100.0% (3/3) |
| | | Towne | 25 mg/mL | 100.0% (3/3) |
| 14 | Nystatin | AD169 | 1,727 U/mL | 100.0% (3/3) |
| | | Towne | 1,727 U/mL | 100.0% (3/3) |
| 15 | Prednisone | AD169 | 0.0003 mg/mL | 100.0% (3/3) |
| | | Towne | 0.0003 mg/mL | 100.0% (3/3) |
| 16 | White Blood Cell | AD169 | 10% (v/v) | 100.0% (3/3) |
| | | Towne | 10% (v/v) | 100.0% (3/3) |
| 17 | Whole blood | AD169 | 10% (v/v) | 100.0% (3/3) |
| | | Towne | 10% (v/v) | 100.0% (3/3) |

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Table 3b. Simplexa Congenital CMV Direct Interference for urine

| No. | Organism | CMV Strain | Tested Concentration | % Agreement*
(# Expected Results/# Tested) |
| --- | --- | --- | --- | --- |
| 1 | Baby powder | AD169 | 10% (w/v) | 100% (3/3) |
| | | Towne | 10% (w/v) | 100% (3/3) |
| 2 | Johnson's Baby Oil (Mineral Oil) | AD169 | 10% (v/v) | 100% (3/3) |
| | | Towne | 10% (v/v) | 100% (3/3) |
| 3 | Meconium | AD169 | 0.5% (w/v) | 100% (3/3) |
| | | Towne | 1% (w/v) | 100% (3/3) |
| 4 | Moist
Towelettes with
Benzalkonium
Chloride | AD169 | 10% (w/v) | 100% (3/3) |
| | | Towne | 10% (w/v) | 100% (3/3) |
| 5 | Nystatin | AD169 | 0.3mg/mL | 100% (3/3) |
| | | Towne | 0.3mg/mL | 100% (3/3) |
| 6 | Stool | AD169 | 2% (w/v) | 100% (3/3) |
| | | Towne | 2% (w/v) | 100% (3/3) |
| 7 | Whole blood | AD169 | 5% (v/v) | 100% (3/3) |
| | | Towne | 10% (v/v) | 100% (3/3) |

4. Assay Reportable Range:
NA

5. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods):
Sample Stability (Fresh vs. Frozen)
The sample stability study for the Candidate Device is comprised of six (6) studies;
a. Sample Stability (Fresh vs Frozen) Saliva Swabs in 3 mL Universal Transport Media (UTM)
b. Sample Stability (Fresh vs Frozen) Saliva Swab in 1 mL Universal Transport Media (UTM), 3 mL Remel M4 RT, 3 mL Remel M4 and 3 mL Remel M6 media
c. Urine Stability (Fresh vs Frozen)
d. Sample Stability Fresh vs Two (2) Freeze-Thaw Cycles 3 mL Universal Transport Medium
e. Sample Stability Fresh vs Two (2) Freeze-Thaw Cycles Saliva Swab in 3 mL Remel M4 and 3 mL Remel M6 media
f. Sample Stability Fresh vs Two (2) Freeze-Thaw Cycles
Results: For all of the tested time points and concentrations there was 100% detection with comparable average Ct values, SD, and %CV observed among the different storage conditions.

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Kit Stability

The Candidate Device and Candidate Device Positive Control Packs were tested for stability. The Candidate Device and Candidate Positive Control Pack is stable for nine (9) months to date as evidenced by real-time stability (on-going) and the Candidate Device Reaction Mix and Positive Control is stable for thirty (30) minutes post thawing.

Positive Control

An email from Diasorin on 6/29/2021 described the positive control as follows. "The inactivated CMV viral stock is in a matrix (true EDTA plasma). This raw material is calibrated using the 1st WHO International Standard (09/162). It is purchased from a vendor (Exact Diagnostics). The CofA is attached. The Inactivated viral stock is diluted in UTM to make the Positive Control at 1 x 10⁵ IU/mL*

*IU/mL is international units (IU) per milliliter (mL)."

6. Detection Limit:

The limit of detection (LoD) was determined for the Simplexa Congenital CMV Direct assay using quantified stocks of three (3) CMV strains (AD169, Towne and Merlin) serially diluted in negative human saliva swab and urine matrices. The LoD for each matrix is presented in Tables 4a and 4b.

Table 4a. Simplexa Congenital CMV Direct Limit of Detection – Saliva Swab

| CMV strain | LoD – Saliva in 1mL UTM | | LoD – Saliva in 3mL UTM | | LoD – Saliva in 3mL M4RT | |
| --- | --- | --- | --- | --- | --- | --- |
| | Copies/mL in saliva | Copies/mL in UTM | Copies/mL in saliva | Copies/mL in UTM | Copies/mL in saliva | Copies/mL in M4RT |
| AD-169 | 6,750 | 500 | 19,250 | 500 | 19,250 | 500 |
| Towne | 6,750 | 500 | 19,250 | 500 | 19,250 | 500 |
| CMV strain | IU/mL in saliva | IU/mL in UTM | IU/mL in saliva | IU/mL in UTM | IU/mL in saliva | IU/mL in M4RT |
| Merlin | 6,750 | 500 | 19,250 | 500 | 19,250 | 500 |

Table 4b. Simplexa Congenital CMV Direct Limit of Detection – Urine

| CMV strain | Copies /mL |
| --- | --- |
| AD-169 | 400 Copies/mL |
| Towne | 800 Copies/mL |
| CMV strain | IU/mL |
| Merlin | 6,400 IU/mL |

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7. Assay Cut-Off:

The initial cutoff study included 288 NTC replicates run on 6 LIAISON MDX instruments at the default assay setting (threshold settings) with no established Ct cutoff with PCR cycles set to run out to 52 cycles. Based on the results of the baseline/default assay runs, the target Ct cutoff was finally set at 42 PCR cycles corresponding to the end of the PCR protocol. However, following the clinical study the cutoff was re-examined and a cutoff of 37.5 was instituted in order to maximize sensitivity and specificity of the assay.

8. Accuracy (Instrument):

NA

9. Carry-Over:

Amplification carry-over for the Simplexa assays has been assessed. The study was designed by alternately placing high positive and negative samples on each disc. No evidence of carry-over contamination was observed.

B Comparison Studies:

1. Method Comparison with Predicate Device:

The performance of the Simplexa Congenital CMV Direct assay was established in a clinical study that included two (2) cohorts based on sample status. Specifically, prospective and retrospective (pre-selected positive and negative samples based on routine laboratory results) samples from infants less than twenty-one (21) days of age, were tested in the clinical agreement study.

Retrospective Study

A total of three hundred forty-six (346) retrospective specimens were collected during the clinical study. Specimens were enrolled, aliquoted and shipped to a central site where they were distributed for Simplex Congenital CMV Direct testing at three (3) laboratories. One (1) central laboratory performed both comparator PCR/bi-directional sequencing assays for the two (2) part Composite Reference Method (CRM). The Composite Reference Method (CRM) utilized two (2) validated PCR followed by bi- directional sequencing assays. A sample had a final sequencing result of 'Detected' if one or both sequencing results were 'Detected'. Conversely a sample had a final sequencing result of 'Not Detected' if both results were 'Not Detected'.

Of the three hundred forty-six (346) specimens tested, one hundred seventy (170) results were generated for saliva swab specimens and one hundred seventy-three (173) results were generated for urine specimens. The results are presented in Tables 5a and 5b.

The saliva swab study included one hundred seventy-three (173) specimens in the analysis. Three (3) specimens were removed from analysis. The three (3) samples yielded an indeterminate final result with the Composite Reference Method (CRM) CMV-2 PCR/Bi-directional sequencing assay preventing the algorithm from producing a result for use as a comparator.

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Table 5a. Simplexa Congenital CMV Direct Clinical Agreement – Saliva Swab (Retrospective)

| Clinical Agreement | | | | PPA | NPA |
| --- | --- | --- | --- | --- | --- |
| Simplexa Congenital CMV Direct Results | Composite Reference Method (CRM) | | | | |
| | Detected | Not Detected | Total | | |
| Detected | 53 | 0 | 53 | 100.0%
(53/53)
95% CI: 93% - 100% | 100.0%
(117/117)
95% CI: 97% - 100% |
| Not Detected | 0 | 117 | 117 | | |
| Total | 53 | 117 | 170 | | |

PPA = Positive Percent Agreement, NPA = Negative Percent Agreement. 95% CI = 95% Confidence Interval<br/>The 95% confidence intervals (CI) were calculated following the Wilson Score method.

Table 5b. Simplexa Congenital CMV Direct Clinical Agreement – Urine (Retrospective)

| Clinical Agreement | | | | PPA | NPA |
| --- | --- | --- | --- | --- | --- |
| Simplexa Congenital CMV Direct Results | Composite Reference Method (CRM) | | | | |
| | Detected | Not Detected | Total | | |
| Detected | 49 | 2a | 51 | 100.0%
(49/49)
95% CI: 93% - 100% | 98.4%
(122/124)
95% CI: 94% -100% |
| Not Detected | 0 | 122 | 122 | | |
| Total | 49 | 124 | 173 | | |

* Two (2) urine samples were positive by routine methodology.<br/>PPA = Positive Percent Agreement, NPA = Negative Percent Agreement. 95% CI = 95% Confidence Interval<br/>The 95% confidence intervals (CI) were calculated following the Wilson Score method.

## Prospective Study

A total of one thousand eight hundred fifty-nine (1,859) saliva swab specimens and one thousand six hundred fifty-six (1,656) urine specimens were prospectively collected as frozen and/or fresh specimens. Of these collected specimens, six (6) saliva swab and thirty-two (32) urine specimens were deemed ineligible and removed from analysis. Specimens were collected from ten (10) collection sites across the USA and two (2) collection sites outside the USA. Testing was performed at six (6) testing sites located in the USA. One (1) central laboratory performed both comparator PCR/bi-directional sequencing assays for the two (2) part Composite Reference Method (CRM).

The Composite Reference Method (CRM) utilized two (2) validated PCR followed by bi-directional sequencing assays. A sample had a final sequencing result of ‘Detected’ if one or both sequencing results were ‘Detected’. Conversely a sample had a final sequencing result of ‘Not Detected’ if both results were ‘Not Detected’.

Prospective clinical agreement was based on a total of one thousand eight hundred fifty-three (1,853) saliva swab specimens and one thousand six hundred twenty-four (1,624) urine specimens. The results are presented in Tables 6a and 6b.

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Table 6a. Simplexa Congenital CMV Direct Clinical Agreement – Saliva Swab (Prospective)

| Clinical Agreement | | | | PPA | NPA |
| --- | --- | --- | --- | --- | --- |
| Simplexa Congenital CMV Direct Results | Composite Reference Method (CRM) | | | | |
| | Detected | Not Detected | Total | | |
| Detected | 16 | 1 | 17 | 94.1%
(16/17)
95% CI: 73% - 99% | 99.9%
(1835/1836)
95% CI: 99.7%-100% |
| Not Detected | 1a | 1835 | 1836 | | |
| Total | 17 | 1836 | 1853 | | |

*One (1) saliva swab specimen was negative by routine methodology.
PPA = Positive Percent Agreement, NPA = Negative Percent Agreement. 95% CI = 95% Confidence Interval
The 95% confidence intervals (CI) were calculated following the Wilson Score method

Table 6b. Simplexa Congenital CMV Direct Clinical Agreement – Urine (Prospective)

| Clinical Agreement | | | | PPA | NPA |
| --- | --- | --- | --- | --- | --- |
| Simplexa Congenital CMV Direct Results | Composite Reference Method (CRM) | | | | |
| | Detected | Not Detected | Total | | |
| Detected | 41 | 0 | 41 | 95.3%
(41/43)
95% CI: 85% - 99% | 100%
(1581/1581)
95% CI: 99.8% - 100% |
| Not Detected | 2a | 1581 | 1583 | | |
| Total | 43 | 1581 | 1624 | | |

*Two (2) urine specimens were negative by routine methodology.
PPA = Positive Percent Agreement, NPA = Negative Percent Agreement.
95% CI = 95% Confidence Interval The 95% confidence intervals (CI)
were calculated following the Wilson Score method.

2. Matrix Comparison:
NA, saliva and urine used as samples

C Clinical Studies:
1. Clinical Sensitivity:
NA
2. Clinical Specificity:
NA

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3. Other Clinical Supportive Data (When 1. and 2. Are Not Applicable):

NA

D Clinical Cut-Off:

NA

E Expected Values/Reference Range:

EXPECTED VALUES

The prevalence of CMV as determined by the Simplexa Congenital CMV Direct assay in a multi-site clinical study with prospectively collected specimens was 0.92% for saliva swabs and 2.52% for urine. Table 7 shows the prevalence of saliva swabs by collection site and Table 2 shows the prevalence of urine by collection site.

Table 7. Prospective Results: Simplexa Congenital CMV Direct Expected Values for Saliva Swabs by Collection Site

| Site ID | Total Specimens | Simplexa congenital CMV Direct Detected | CMV Prevalence |
| --- | --- | --- | --- |
| 7 | 142 | 0 | 0.00% |
| 8 | 16 | 1 | 6.25% |
| 9 | 632 | 3 | 0.47% |
| 10 | 9 | 0 | 0.00% |
| 12 | 14 | 0 | 0.00% |
| 13 | 11 | 1 | 9.09% |
| 14 | 9 | 0 | 0.00% |
| 15 | 10 | 0 | 0.00% |
| 18 | 8 | 0 | 0.00% |
| 19 | 1002 | 12 | 1.20% |
| All | 1853 | 17 | 0.92% |

F Other Supportive Instrument Performance Characteristics Data:

NA

VIII Proposed Labeling:

The labeling supports the finding of substantial equivalence for this device.

IX Conclusion:

The submitted information in this premarket notification is complete and supports a substantial equivalence decision.

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