Predicate Devices

Not Found

Reference Devices

AI/ML (Artificial Intelligence / Machine Learning)

No
The device description focuses on LAMP technology and monitoring absorbance changes, with no mention of AI or ML. The performance studies describe standard clinical trial methodology and statistical analysis, not AI/ML model training or evaluation.

Therapeutic

No.
The device is an in vitro diagnostic test system used to detect CMV DNA as an aid in diagnosing congenital CMV infection, not to treat or cure a disease or condition.

Diagnostic

Yes

The device is explicitly stated as a "qualitative, in vitro diagnostic test system" and is "used as an aid in the diagnosis of congenital CMV infection."

SaMD (Software as a Medical Device)

No

The device description explicitly mentions the "Alethia instrument" which monitors reaction solution absorbance characteristics, indicating a hardware component is integral to the device's function.

IVD (In Vitro Diagnostic)

Yes, this device is an IVD (In Vitro Diagnostic).

Here's why:

Explicitly stated in the Intended Use: The document clearly states, "The Alethia CMV DNA Amplification Assay... is a qualitative, in vitro diagnostic test system..."
Purpose of the device: The device is designed to detect Cytomegalovirus (CMV) DNA in saliva samples, which is a biological specimen taken from the human body. This is a key characteristic of an in vitro diagnostic test.
Used as an aid in diagnosis: The intended use specifies that the test is used "as an aid in the diagnosis of congenital CMV infection." This indicates its role in providing information for clinical decision-making.
Performed on an instrument: The assay is performed on the Alethia instrument, which is a common setup for in vitro diagnostic tests.
External controls: The inclusion of external controls is a standard practice for ensuring the quality and reliability of in vitro diagnostic tests.

All of these points align with the definition and characteristics of an In Vitro Diagnostic device.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

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 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.

Product codes

QDZ

Device Description

The Alethia CMV Assay Test System, including the Alethia CMV DNA Amplification Assay and the Alethia CMV External Controls, is based on loopmediated amplification (LAMP) technology. The assay targets a region of the Cytomegalovirus genome that is conserved across multiple CMV strains. The Alethia CMV target is a 194 base pair (bp) sequence of the Human herpesvirus 5 genome.

LAMP uses specially designed primers to provide for specific and continuous isothermal DNA amplification. A bv-product of amplification is magnesium pyrophosphate, which forms a white precipitate leading to a turbid solution. Reaction solution absorbance characteristics are monitored by the Alethia™ instrument.

Changes in reaction solution turbidity created by precipitation of magnesium pyrophosphate indicate the presence of target DNA. The absence of target DNA results in no significant change in sample absorbance.

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

saliva samples

Indicated Patient Age Range

neonates younger than 21 days of age.

Intended User / Care Setting

Not Found

Description of the training set, sample size, data source, and annotation protocol

Not Found

Description of the test set, sample size, data source, and annotation protocol

The Alethia CMV Assay Test System was evaluated from August 2017 to March 2018 at seven clinical study sites representing geographically distinct regions throughout the United States, Canada, Europe, and Australia. One-thousand fivehundred and fourteen (1,514) specimens were prospectively collected and tested with Alethia CMV assay (forty-seven of these specimens were frozen at ≤ -20℃ after collection and tested later). The saliva swabs were collected at least one hour after breastfeeding.

For estimation of composite reference method (CRM) positive percent agreement, thirty-four (34) archived specimens were also tested by Alethia CMV assay. The archived samples were de-identified samples previously evaluated from prospective clinical studies and found to have CMV infection. These samples were collected from infants less than 21 days of age and stored at -80 ℃ after the completion of the initial testing. The status of breastfeeding time in relation to time of saliva samples collected was not available.

All samples were tested with Alethia CMV Assay Test System at the study sites, then shipped to Meridian Bioscience, Inc. for CRM testing. The CRM consisted of two manufacturer-developed and validated PCR assays. Samples positive by either PCR assay were further tested by bidirectional sequencing (BDS). Samples were considered positive when bi-directional sequencing results from either comparator PCR assay confirmed the presence of CMV DNA. Samples were considered negative when neither of the comparator PCR assays produced amplicon at the end of the 40-cycle amplification or BDS was negative.

PCR #1	PCR #2	Bidirectional Sequencing	Composite Reference Method
+	+	+	+
+	+	-	-
+	-	+	+
+	-	-	-
-	+	+	+
-	+	-	-
-	-	n/a	-

Summary of Performance Studies

Study Type: Clinical Studies (Prospective and Preselected Positive archived sample study)
Sample Size: 1,514 prospectively collected samples (5 positive by CRM, 1,475 negative by CRM, 34 invalid by CRM and removed from analysis). 34 archived preselected positive samples.
Standalone Performance: Not Applicable
Key Results:
Prospective Study results:
Positive Percent Agreement: 100% (5/5) (95% CI: 56.7%; 100%)
Negative Percent Agreement: 99.8% (1,472/1,475) (95% CI: 99.4%; 99.9%)

Preselected Positive archived sample study:
Positive Percent Agreement: 100% (34/34) (95% CI: 89.9%; 100%)
Negative Percent Agreement: Not Applicable

Combined Positive and Negative percent agreements:
Positive Percent Agreement: 100% (39/39) (95% CI: 91.0%; 100%)
Negative Percent Agreement: 99.8% (1,472/1,475) (95% CI: 99.4%; 99.9%)

The rate of initial invalid results was 1.7% (27/1,548). After re-testing, the rate of final invalid results was 0.06% (1/1,548) with 95% CI: 0.01%; 0.37%.

Key Metrics

Positive Percent Agreement: 100% (5/5), 100% (34/34), combined 100% (39/39)
Negative Percent Agreement: 99.8% (1,472/1,475)

Predicate Device(s)

Not Found

Reference Device(s)

K160829, K123423

Predetermined Change Control Plan (PCCP) - All Relevant Information

Not Found

Regulation Number and Section

§ 866.3181 Cytomegalovirus nucleic acid detection device for congenital cytomegalovirus infection.

(a)
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.(b)
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.

Summary

0

EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR Alethia CMV DNA Amplification Assay

DECISION SUMMARY

A. DEN Number:

DEN180040

B. Purpose for Submission:

De Novo request for evaluation of automatic class III designation for the Alethia CMV Assay Test System.

C. Measurands:

Cytomegalovirus (CMV) DNA

D. Type of Test:

Qualitative loop-mediated isothermal DNA amplification (LAMP) technology

E. Applicant:

Meridian Bioscience, Inc.

F. Proprietary and Established Names:

Alethia CMV Assay Test System G. Regulatory Information:

1. Regulation section: 21 CFR 866.3181
1. Classification: Class II (Special Controls)
1. Product code: QDZ
1. Panel: 83 - Microbiology

1

H. Indications For Use:

1. Indications for use:

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 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.

2. Special conditions for use statement(s):

For in vitro diagnostic use only

Prescription use only

3. Special instrument requirements:

Alethia Automated Isothermal Amplification and Detection System

I. Device Description:

The Alethia CMV Assay Test System, including the Alethia CMV DNA Amplification Assay and the Alethia CMV External Controls, is based on loopmediated amplification (LAMP) technology. The assay targets a region of the Cytomegalovirus genome that is conserved across multiple CMV strains. The Alethia CMV target is a 194 base pair (bp) sequence of the Human herpesvirus 5 genome.

LAMP uses specially designed primers to provide for specific and continuous isothermal DNA amplification. A bv-product of amplification is magnesium pyrophosphate, which forms a white precipitate leading to a turbid solution. Reaction solution absorbance characteristics are monitored by the Alethia™ instrument.

2

Changes in reaction solution turbidity created by precipitation of magnesium pyrophosphate indicate the presence of target DNA. The absence of target DNA results in no significant change in sample absorbance.

Reagents/Materials Provided:

The maximum number of tests obtained from this test kit is listed on the outer box.

Alethia CMV Test Device: Two-chambered device containing lyophilized 1. amplification reagents (DNA polymerase, deoxynucleotide triphosphates) with Cytomegalovirus specific primers (TEST Chamber) and human mitochondrial DNA-specific primers (CONTROL Chamber).
1. Alethia CMV Buffer I: Lysis solution containing 0.2N sodium hydroxide and 1% Triton-X 100.
Alethia CMV Buffer II: Tris-buffer solution containing 0.09% azide as a 3. preservative.

Materials Required but Not Provided: Alethia CMV External Control Reagents

J. Standard/Guidance Document Referenced (if applicable):

EP05-A3: Evaluation of Precsion Performance of Quantitative Measurement Procedures.

EP12-A2: User Protocol for Evaluation of Qualitative Test Performance.

EP17-A2: Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures.

EP07-A3: Interference Testing in Clinical Chemistry. FDA Recognition Number 7-127.

MM03-3rd Edition: Molecular Diagnostic Methods for Infectious Diseases; Approved Guideline.

K. Test Principle:

The Alethia CMV DNA Amplificaiton assay contains one lyophilized amplification reagent bead in each of two chambers: a TEST chamber with Cytomegalovirus specific primers and a CONTROL chamber with human mitochondrial DNA-specific primers. Human mitochondrial DNA in saliva samples, and the human mitochondrial DNAspecific primers in the Test Device CONTROL chamber, functions as the Internal Control for the assay. During specimen preparation, human mitochondrial DNA is liberated with the Cytomegalovirus DNA to allow for parallel processing of target DNA and Control DNA through amplification and detection. The Internal Control monitors DNA amplification inhibition, assay reagent performance, and sample processing effectiveness. The Control target must be amplified and detected in the final reaction or the test is considered invalid and results not reported.

3

The Alethia instrument monitors changes in absorbance characteristics by measuring transmission of light through the Test and Control reaction solutions. Light transmission is checked at the assay Run Start (Signalinitial, S;) and at the assay Run End (Signalfinal, St). The Alethia instrument calculates the change in light transmission between Run End and Run Start (Sr:S:) and compares the ratio to a fixed cut-off value.

Fixed cut-off values for the TEST chamber are used to report sample assay results. TEST chamber Sf:Si ratios less than 82% are reported as 'POSITIVE'. TEST chamber Sf:Si ratios greater than or equal to 82% are reported as 'NEGATIVE'. Numerical values are not reported.

Fixed cut-off values for the CONTROL chamber are used to determine validity. CONTROL chamber Sr:S; ratios less than 90% are considered valid and allow for reporting of TEST chamber results (POSITIVE or NEGATIVE). CONTROL chamber Sf:Si ratios greater than or equal to 90% are considered invalid and prevent reporting of TEST chamber results. Invalid CONTROL chamber reactions are reported as 'INVALID'. Numerical values are not reported.

More stringent cut-off criteria are applied to the CONTROL chamber reaction to ensure amplification is not inhibited, reagents are performing as intended, and that sample processing was performed appropriately.

| Sample
ID | Reported
Result | Interpretation |
|---------------------|--------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Patient
Specimen | POSITIVE | Cytomegalovirus target DNA was detected |
| | NEGATIVE | No Cytomegalovirus DNA detected |
| | INVALID* | No reportable result. The test should be repeated:
Samples can be retested from the Buffer II preparation within 3 hours of
sample preparation. |
| Positive
Control | POSITIVE | Valid positive control result. Reagents active at time of use; Alethia
instrument performing correctly. |
| | NEGATIVE | Incorrect control result. Repeat the control tests as the first step in
determining the root cause of the failure. If control failures are repeated
please contact Meridian's Technical Services at
1-800-343-3858 (US) or your local distributor. |
| | INVALID | No reportable result. Run must be repeated.
Improper sample preparation, reagent failure, instrument failure or
internal control failure. |
| Negative
Control | POSITIVE | Incorrect control result. Repeat the control tests as the first step in
determining the root cause of the failure. If control failures are repeated
please contact Meridian's Technical Services at
1-800-343-3858 (US) or your local distributor. |

Interpretation Of Results

4

| | NEGATIVE | Valid negative control result. Reagents active at time of use, Alethia
instrument performing correctly. |
|---------------|----------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| | INVALID | No reportable result. Run must be repeated.
Improper sample preparation, reagent failure, instrument failure or
internal control failure. |
| EMPTY
WELL | NONE | No Alethia Test Device in the Alethia instrument well.
OR
The Alethia Test Device present is compromised due to sample
preparation failure, dirty device or improperly seated device. Repeat
testing on the sample. |

*Interpretation Notes

· Invalid results may occur as a result of inhibitory specimens, improper sample preparation, reagent failure, no human DNA in the specimen, instrument failure, or internal control failure.
· For VTM specimens, retesting may be performed with the original specimen if sufficient volume remains (see Specimen Collection and Preparation section for additional guidance).

L. Performance Characteristics:

1. Analytical performance:

a. Precision/Reproducibility:

Panels of two sample types, dry swab and VTM, were supplied to three laboratories for this reproducibility study. The panels included contrived CMV samples manufactured as moderatepositive, low-positive samples, and high-negative samples (30 replicates per site for each sample). The panel also included one true negative sample (10 replicates per site). Positive and Negative Controls were tested with each panel also (10 replicates per site). Testing was performed by different operators at each site on the same day for five days. Three lots of Alethia CMV kits and six Alethia instruments were used in this study. Mean values, repeatability, between-operator, between-day, and between-site components of variance for numeric values of Sr.Si ratios, percent of positive and negative results are provided in the tables below for each sample type tested in the reproducibility study.

Saliva Samples on Dry Swabs
			Repeatability		Between-
operators1		Between-
day		Between-
site2		Reproducibility		%Pos.	%Neg results
	N	Mean	SD	%CV	SD	%CV	SD	%CV	SD	%CV	SD	%CV	results
Moderate
Positive	90	62.26	2.93	4.7%	1.69	2.7%	0.00	0.0%	0.91	1.5%	3.50	5.6%	100%	0%
Low
Positive	90	62.38	4.44	7.1%	1.96	3.1%	1.11	1.8%	0.00	0.0%	4.98	8.0%	98.9%	1.1%
High
Negative	90	99.05	6.68	6.7%	0.97	1.0%	0.00	0.0%	0.23	0.2%	6.76	6.8%	3.3%	96.7%
True	30	100.13	0.69	0.7%	n/a	n/a	0.31	0.3%	0.40	0.4%	0.86	0.9%	0%	100%

Reproducibility for Dry Swab Samples

5

Negative3
Positive
Control3	30	61.84	2.72	4.4%	n/a	n/a	0.00	0.0%	1.11	1.8%	2.94	4.8%	100%	0%
Negative
Control3	30	100.23	2.06	2.1%	n/a	n/a	0.17	0.2%	0.22	0.2%	2.08	2.1%	0%	100%

1 Includes between-operator and between-instrument components

2 Includes between-site and between-kit lot components

3 Samples were run at each site for 5 days with 2 runs per day and 1 replicate per run.

6

Reproducibility for VTM samples

	Saliva Samples in VTM
			Repeatability		Between-
operators1		Between-
day		Between-
site2		Reproducibility		% Pos	% Neg
	N	Mean	SD	%CV	SD	%CV	SD	%CV	SD	%CV	SD	%CV	results	results
Moderate
Positive	90	61.99	3.18	5.1%	1.02	1.6%	0.00	0.0%	1.20	1.9%	3.55	5.7%	100%	0%
Low Positive	90	61.71	2.37	3.8%	1.43	2.3%	0.00	0.0%	0.67	1.1%	2.85	4.6%	100%	0%
High Negative	90	100.11	4.82	4.8%	0.00	0.0%	0.74	0.7%	0.00	0.0%	4.88	4.9%	98.9%	1.1%
True Negative3	30	99.20	7.71	7.8%	N/A	N/A	0.00	0.0%	0.00	0.0%	7.71	7.8%	96.7%	3.3%
Positive
Control3	30	62.22	2.39	3.8%	N/A	N/A	0.00	0.0%	0.49	0.8%	2.44	3.9%	100%	0%
Negative
Control3	30	100.30	2.02	2.0%	N/A	N/A	0.00	0.0%	0.12	0.1%	2.02	2.0%	0%	100%

1 Includes between-operator and between-instrument components

2 Includes between-site and between-kit lot components

3 Samples were run at each site for 5 days with 2 runs per day and 1 replicate per run.

Within-Laboratory Precision Study

Panels of two sample types, dry swab and VTM, were tested at one site (internal) over 6 days. The panels included contrived CMV samples manufactured as moderate-positive, low-positive (around 2X LoD), and high-negative samples. The panel also included one true negative sample, Positive and Negative Controls. Three kit lots were used during the study, one lot per day of testing. Each kit lot was tested twice over the 6-day testing period. Mean values, repeatability, between-operator, between-day, and between-kit lot components of variance for numeric values of Sr.S; ratios, percent of positive and negative results are provided in the tables below for each sample type tested in the precision study.

Within-Laboratory Precision for Dry Swab Samples

| | | | Repeatability1 | | Between-
operators1 | | Between-
day | | Between-
kit lot | | Reproducibility | | %Pos
results | %Neg results | |
|-----------------------------|----|-------|----------------|------|------------------------|------|-----------------|------|---------------------|------|-----------------|------|-----------------|--------------|--|
| Saliva Samples on Dry Swabs | | | | | | | | | | | | | | | |
| | N | Mean | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | | | |
| Moderate
Positive | 48 | 60.29 | 1.99 | 3.3% | 0.00 | 0.0% | 0.00 | 0.0% | 0.74 | 1.2% | 2.12 | 3.5% | 100% | 0% | |
| Low
Positive | 48 | 60.23 | 1.70 | 2.8% | 0.62 | 1.0% | 0.00 | 0.0% | 0.74 | 1.2% | 1.95 | 3.2% | 100% | 0% | |
| High
Negative | 48 | 98.26 | 6.46 | 6.6% | 4.36 | 4.4% | 1.42 | 1.4% | 0.80 | 0.8% | 7.96 | 8.1% | 4.2% | 95.8% | |
| True
Negative | 48 | 99.94 | 0.61 | 0.6% | 0.00 | 0.0% | 0.12 | 0.1% | 0.14 | 0.1% | 0.64 | 0.6% | 0% | 100% | |
| Positive
Control | 24 | 60.57 | 1.96 | 3.2% | 0.00 | 0.0% | 1.47 | 2.4% | 0.00 | 0.0% | 2.45 | 4.0% | 100% | 0% | |
| Negative
Control | 24 | 99.73 | 0.29 | 0.3% | 0.00 | 0.0% | 0.18 | 0.2% | 0.16 | 0.2% | 0.38 | 0.4% | 0% | 100% | |

1 Includes between-operator and between-instrument components

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Saliva Samples in VTM
	N	Mean	Repeatability		Between-
operators1		Between-
day		Between-
kit lot		Reproducibility		%Pos results	%Neg results
			SD	%CV	SD	%CV	SD	%CV	SD	%CV	SD	%CV
Moderate
Positive	48	60.62	1.36	2.2%	0.91	1.5%	0.00	0.0%	0.77	1.3%	1.81	3.0%	100%	0%
Low
Positive	48	60.55	1.67	2.8%	0.89	1.5%	0.72	1.2%	0.00	0.0%	2.02	3.3%	100%	0%
High
Negative	48	99.91	0.64	0.6%	0.00	0.0%	0.22	0.2%	0.00	0.0%	0.68	0.7%	0%	100%
True
Negative	48	100.00	0.84	0.8%	0.28	0.3%	0.00	0.0%	0.00	0.0%	0.89	0.9%	0%	100%
Positive
Control	24	60.28	2.12	3.5%	0.00	0.0%	1.09	1.8%	0.00	0.0%	2.38	4.0%	100%	0%
Negative
Control	24	100.29	1.53	1.5%	0.28	0.3%	0.00	0.0%	0.00	0.0%	1.56	1.6%	0%	100%

Within-Laboratory Precision for VTM Samples

1 Includes between-operator and between-instrument components

8

b. Linearity/assay reportable range:

Not Applicable

c. Traceability, Stability, Expected values (controls, calibrators, or methods):

The stability of the Alethia CMV Assay Test System, including the Alethia CMV External Controls was evaluated in real-time studies, to support the proposed expiration dating period of 15 months for the Alethia CMV DNA Amplification Assay and the Alethia CMV External Controls. Three lots of Assay and External Control reagent components were used and the results obtained for the Alethia CMV DNA Amplification Assay and the Alethia CMV External Controls support expiration dating periods of 15 months for both the assay reagents and the external controls.

d. Detection limit:

The analytical sensitivity, described as Limit of Detection (LoD), is the concentration at which the Alethia CMV Assay Test System has positive results at least 95% of the time. The LoD of the Alethia CMV assay was determined for Cytomegalovirus strain Merlin in a negative sample matrix. Additionally saliva samples collected from CMV negative healthy adults were tested and confirmed negative by the Alethia CMV assay. Three kit lots of Alethia CMV assay reagents and eight Alethia instruments were used. A minimum of 6 dilutions with 20 replicates were tested for each lot. LoD was determined using Probit analysis. LoD was determined separately for the two sample types, dry swab and swab in VTM. LoD concentrations for each sample type are summarized below.

| Sample
Type | LoD
(Copies/mL) |
|-----------------|--------------------|
| Dry Swab1 | 1,025 |
| Swab in
VTM2 | 15,686 |

1The CMV cp/mL concentration in saliva on the swab for dry swab samples is calculated by multiplying the CMV concentration in Buffer I by a factor of 4.75 (the 4.75-fold dilution occurs when 0.080 mL of saliva on the swab is added to 0.3 mL of Buffer I [380÷80 = 4.75]; 100% transfer is assumed).

2The CMV cp/mL concentration in saliva on the swab for VTM samples is calculated by multiplying the CMV concentration in VTM by a factor of 13.5 (the 13.5-fold dilution occurs when 0.080 mL of saliva on the swab is placed in 1 mL of VTM [1080=80 = 13.5]; 100% transfer is assumed).

Assay Inclusivity

The inclusivivty of the Alethia CMV Assay Test System was evaluated by testing samples containing 3 additional CMV strains other than CMV strain Merlin. These strains include Toledo. Towne, and AD-169. Ouantified strains were diluted in simulated negative clinical matrix to approximately 2-3X LoD for both VTM and dry swab and

9

tested in triplicate. All strains tested for both sample types produced positive results with Alethia CMV.

| CMV
Strain

Tested	Dry Swab Samples		VTM Swab Samples
	Test
concentration
(copies/mL)	Test Results
(npos/ntotal)	Test
concentration
(copies/mL)	Test Results
(npos/ntotal)
AD-169	2,493	3/3	45,077	3/3
Toledo	2,493	3/3	45,077	3/3
Towne	2,493	3/3	45,077	3/3

e. Analytical specificity:

Cross Reactivity Studies

Cross-reactivity studies were performed with a panel of 40 microorganisms and human genomic DNA, each diluted in dry swab simulated negative clinical matrix. Microorganims selected include those with genetic similarity to CMV and those likely to be present in the oral cavity of neonates. Microorganims were diluted in dry swab simulated negative clinical matrix to the indicated concentration (see table below) and tested in triplicate. No cross-reactivity with the Alethia CMV assay was observed.

Microorganism	Test concentration	Microorganism	Test concentration
Acinetobacter baumannii	1.2x107 CFU/mL	Adenovirus	3.80x105 TCID50/mL
Actinomyces odontolyticus	1.2x107 CFU/mL	Coronavirus	2.19x105 TCID50/mL
Bordetella pertussis	1.2x107 CFU/mL	Coxsackievirus	4.07x106 TCID50/mL
Candida albicans	1.2x107 CFU/mL	Enterovirus 71	1.26x105 TCID50/mL
Escherichia coli ATCC 35218	1.2x107 CFU/mL	Epstein Barr Virus	3.39x108 cp/mL
Fusobacterium nucleatum	1.2x107 CFU/mL	Herpes Simplex Virus 1	9.5x105 TCID50/mL
Haemophilus influenzae	1.2x107 CFU/mL	Herpes Simplex Virus 2	1.3x105 TCID50/mL
Haemophilus parainfluenzae	1.2x107 CFU/mL	Human herpesvirus 6B	6.16x107 cp/mL
Moraxella catarrhalis	1.2x107 CFU/mL	Human herpesvirus 7	3.80x105 TCID50/mL
Mycoplasma pneumoniae	3.70x107 CCU/mL	Human herpesvirus 8	2.13x108 cp/mL

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Porphyromonas gingivalis	$1.2x10^7$ CFU/mL	Human metapneumovirus	$6.61x10^5$ TCID50/mL
Pseudomonas aeruginosa	$1.2x10^7$ CFU/mL	Influenza A	$3.80x10^5$ TCID50/mL
Staphylococcus aureus	$1.2x10^7$ CFU/mL	Influenza B	$4.57x10^5$ TCID50/mL
Staphylococcus epidermidis	$1.2x10^7$ CFU/mL	Parainfluenza virus 1	$1.95x10^6$ TCID50/mL
Streptococcus agalactiae (GBS)	$1.2x10^7$ CFU/mL	Parainfluenza virus 2	$5.89x10^6$ TCID50/mL
Streptococcus anginosus (Group F)	$1.2x10^7$ CFU/mL	Parainfluenza virus 3	$2.19x10^5$ TCID50/mL
Streptococcus mitis	$1.2x10^7$ CFU/mL	Respiratory syncytial virus A	$3.2x10^5$ TCID50/mL
Streptococcus oralis	$1.2x10^7$ CFU/mL	Respiratory syncytial virus B	$4.6x10^5$ TCID50/mL
Streptococcus salivarius	$1.2x10^7$ CFU/mL	Rhinovirus	$1.51x10^5$ TCID50/mL
Streptococcus sanguinis	$1.2x10^7$ CFU/mL	Varicella Zoster Virus	$3.36x10^9$ cp/mL
Human Genomic DNA	$6.18x10^6$ cp/mL	n/a	n/a

Microbial Interference

Microbial interference studies were performed with a panel of 40 microorganisms and human genomic DNA, each diluted in dry swab simulated positive clinical matrix. Each sample tested contained CMV at a concentration of 3X LoD and microorganism or human genomic DNA at the test concentration indicated above (see table in Crossreactivity section for microorganisms tested and final test concentrations). Once prepared, each sample was tested in triplicate. No microbial interference with the Alethia CMV DNA Amplification Assay was observed (i.e., all microorganisms diluted in dry swab simulated positive clinical matrix produced 3/3 positive test results with the Alethia CMV assay).

Interfering Substances

Interference testing was performed in the presence of chemical and biological substances introduced directly into contrived CMV low positive (3X LoD) and negative samples. Two unique positive samples and one negative sample were tested in triplicate. The substances tested, the concentrations evaluated, and test results are shown in the following table. No interference was observed with the following substances with the Alethia CMV Assay Test System at the specified test concentrations (i.e., all positive replicates tested produced positive Alethia CMV test results and all negative replicates tested produced negative Alethia CMV test results).

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		Test Results (ndetected/ntotal)
Substance Tested	Test
concentration	CMV
Negative
Sample	CMV
Positive
Sample #1	CMV
Positive
Sample #2
Infants' Pain & Fever
(Acetaminophen)	0.2 mg/mL	0/3	3/3	3/3
Acetylsalicylic acid	0.65 mg/mL	0/3	3/3	3/3
Caffeine	0.06 mg/mL	0/3	3/3	3/3
EnfamilTM Fer-In-Sol®
(Ferrous Sulfate)	1.5 mg/mL	0/3	3/3	3/3
Enfamil Premium®
Infant Formula
Newborn	10% v/v	0/3	3/3	3/3
Infants' Mylicon® Gas
Relief (Simethicone)	2 mg/0.3 mL	0/3	3/3	3/3
Gaviscon® infant
(Sodium alginate)	1.2 mg/mL	0/3	3/3	3/3
Magnesium alginate	0.467 mg/mL	0/3	3/3	3/3
Infants' Ibuprofen	0.5 mg/mL	0/3	3/3	3/3
EnfamilTM Poly-Vi-Sol®	8% v/v	0/3	3/3	3/3
Little Remedies® Saline
spray/drops	10% v/v	0/3	3/3	3/3
Methadone	0.002 mg/mL	0/3	3/3	3/3
Morphine sulphate	0.0005
mg/mL	0/3	3/3	3/3
Nystatin	1000 U/mL	0/3	3/3	3/3
Prednisone	0.0003	0/3	3/3	3/3
	mg/mL
Casein	10 mg/mL	0/3	3/3	3/3
Mucin*	25 mg/mL	0/3	3/3	3/3
White blood cells	10% v/v	0/3	3/3	3/3
Whole blood	10% v/v	0/3	3/3	3/3

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When mucin was tested at a concentratin of 50 mg/mL, the negative sample tested negative in 3/3 replicates and CMV positive sample #1 tested positive in 3/3 replicates. CMV positive sample #2 produced two invalid test results, and one positive test result for the three replicates tested. The mucin concentration in the samples was reduced to 25 mg/mL and testing was repeated, producing the results shown in the table. No interference was observed with a mucin concentration of 25 mg/mL. A limitation was added to the device labeling to mitigate this finding.

f. Sample Stability:

Studies were performed to assess both sample stability and sample freeze-thaw stability.

For sample stability, a sample panel was prepared by spiking quantified CMV virus into simulated dry swab clinical matrix, BD UVT, and Puritan UniTranz-RT transport media. Negative samples and positive samples (3X LoD) of each type were prepared. Each (b)(4)

Results support that saliva swabs may be stored at room temperature (19-30 ℃), refrigerated (2-8 ℃ ), or frozen (