BioCode Respiratory Pathogen Panel (RPP)

K192485 · Applied BioCode, Inc. · OCC · Dec 23, 2019 · Microbiology

Device Facts

Record IDK192485
Device NameBioCode Respiratory Pathogen Panel (RPP)
ApplicantApplied BioCode, Inc.
Product CodeOCC · Microbiology
Decision DateDec 23, 2019
DecisionSESE
Submission TypeTraditional
Regulation21 CFR 866.3980
Device ClassClass 2
AttributesReal-World Evidence, Pediatric

Real-World Evidence

SubmissionDeviceSponsorRWD SourcesRWE Use SummaryKey Tags
K192485 · Dec 23, 2019BioCode Respiratory Pathogen Panel (RPP)Applied BioCode, Inc.Archived clinical nasopharyngeal swab (NPS) specimensRetrospective clinical study using archived specimens to supplement prospective clinical study data for pathogens with low prevalence.Retrospective study; Archived clinical specimens; Clinical performance

Clinical Evidence

Study DesignPopulationComparatorKey Endpoints
Retrospective evaluation of preselected archived clinical specimensPatients suspected of respiratory tract infections; Sample Size: 165; Number of Sites: 5FDA-cleared molecular multiplexed respiratory pathogen panelPositive and negative percent agreement

Indications for Use

The BioCode® Respiratory Pathogen Panel (RPP) is a qualitative multiplexed nucleic acid-based in vitro diagnostic test intended for use with BioCode MDx-3000 Instrument. The BioCode RPP is capable of the simultaneous detection and identification of nucleic acids from multiple viruses and bacteria extracted from nasopharyngeal swab (NPS) samples obtained from individuals with signs and/or symptoms of respiratory tract infection. The following pathogens and subtypes are identified using the BioCode RPP: Adenovirus, Coronavirus (229E, OC43, HKU1, and NL63), Human Metapneumovirus A/B, Influenza A, including subtypes H1, H1 2009 Pandemic, and H3, Influenza B, Parainfluenza 1, Parainfluenza 2, Parainfluenza 3, Parainfluenza 4, Respiratory Syncytial Virus A/B, Rhinovirus/Enterovirus, Bordetella pertussis, Chlamydia pneumoniae, Mycoplasma pneumoniae. The detection and identification of specific viral and bacterial nucleic acids from individuals exhibiting signs and/or symptoms of a respiratory infection aids in the diagnosis of respiratory infection if used in conjunction with other clinical and epidemiological information. The results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. Negative results in the setting of a respiratory illness may be due to infection with pathogens that are not detected by this test, or lower respiratory tract infection that may not be detected by a nasopharyngeal swab specimen. Positive results do not rule out co-infection with other organisms: the agent(s) detected by the BioCode RPP may not be the definite cause of disease. Additional laboratory testing (e.g. bacterial and viral culture, immunofluorescence, and radiography) may be necessary when evaluating a patient with possible respiratory tract infection.

Device Story

BioCode RPP is a multiplexed nucleic acid-based in vitro diagnostic test for respiratory pathogens; used with BioCode MDx-3000 system. Input: nasopharyngeal swab (NPS) samples in transport media. Process: automated nucleic acid extraction (NucliSens easyMAG or Roche MagNA Pure 96); PCR amplification; target capture; signal generation; optical detection using barcoded magnetic beads (BMB) capturing biotinylated products with streptavidin conjugate. Output: qualitative detection/identification of 17 viral/bacterial targets. Used in clinical laboratories by trained personnel. Results aid diagnosis of respiratory infections; must be used with other clinical/epidemiological data. Benefits: simultaneous detection of multiple pathogens from single specimen; aids clinical decision-making for patient management.

Clinical Evidence

Prospective clinical study (n=2649) compared BioCode RPP to FDA-cleared molecular multiplex panel. Overall validity rate 99.9%. PPA/NPA calculated for 17 analytes. Performance stratified by site and extraction method (NucliSENS easyMAG/MagNA Pure 96). Retrospective study (n=165) and contrived specimen study (n=110) supplemented data for rare pathogens. No significant performance differences observed between extraction methods.

Technological Characteristics

Multiplex nucleic acid assay; uses barcoded magnetic beads (BMBs) for target capture; biotinylated PCR products; SA-PE fluorescence detection. Automated processing on BioCode MDx-3000. Compatible with NucliSENS easyMAG or MagNA Pure 96 extraction. 96-well plate format. Software-based automated interpretation of MFI thresholds.

Indications for Use

Indicated for individuals with signs/symptoms of respiratory tract infection. Used for qualitative detection/identification of 17 viral and bacterial pathogens/subtypes from nasopharyngeal swab (NPS) samples. Not for sole diagnostic use; results must be used with clinical/epidemiological data. Cannot differentiate Rhinovirus/Enterovirus.

Regulatory Classification

Identification

A respiratory viral panel multiplex nucleic acid assay is a qualitative in vitro diagnostic device intended to simultaneously detect and identify multiple viral nucleic acids extracted from human respiratory specimens or viral culture. The detection and identification of a specific viral nucleic acid from individuals exhibiting signs and symptoms of respiratory infection aids in the diagnosis of respiratory viral infection when used in conjunction with other clinical and laboratory findings. The device is intended for detection and identification of a combination of the following viruses:(1) Influenza A and Influenza B; (2) Influenza A subtype H1 and Influenza A subtype H3; (3) Respiratory Syncytial Virus subtype A and Respiratory Syncytial Virus subtype B; (4) Parainfluenza 1, Parainfluenza 2, and Parainfluenza 3 virus; (5) Human Metapneumovirus; (6) Rhinovirus; and (7) Adenovirus.

Special Controls

*Classification.* Class II (special controls). The special controls are:(1) FDA's guidance document entitled “Class II Special Controls Guidance Document: Respiratory Viral Panel Multiplex Nucleic Acid Assay;” (2) For a device that detects and identifies Human Metapneumovirus, FDA's guidance document entitled “Class II Special Controls Guidance Document: Testing for Human Metapneumovirus (hMPV) Using Nucleic Acid Assays;” and (3) For a device that detects and differentiates Influenza A subtype H1 and subtype H3, FDA's guidance document entitled “Class II Special Controls Guidance Document: Testing for Detection and Differentiation of Influenza A Virus Subtypes Using Multiplex Nucleic Acid Assays.” See § 866.1(e) for the availability of these guidance documents.

Predicate Devices

Submission Summary (Full Text)

{0} Food and Drug Administration 10903 New Hampshire Avenue Silver Spring, MD 20993-0002 www.fda.gov ### 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY AND INSTRUMENT # **I Background Information:** # **A 510(k) Number** K192485 # **B Applicant** Applied BioCode, Inc. # **C Proprietary and Established Names** BioCode Respiratory Pathogen Panel (RPP) # **D Regulatory Information** | Product Code(s) | Classification | Regulation Section | Panel | | --- | --- | --- | --- | | OCC, OZE, OEP, OEM, OOU, OTG, OZX, OZY, OZZ, NSU | Class II | 21 CFR 866.3980 - Respiratory Viral Panel Multiplex Nucleic Acid Assay | MI - Microbiology | # **II Submission/Device Overview:** # **A Purpose for Submission:** New device # **B Measurand:** Adenovirus, Human Metapneumovirus (hMPV) A/B, Influenza A (Flu A), Influenza A subtype H1 (Flu A/H1), Influenza A subtype H1 2009 Pandemic (Flu A/H1pdm09), Influenza A subtype H3 (Flu A/H3), Influenza B (Flu B), Coronavirus (229E, HKU1, OC43, and NL63), Parainfluenza virus 1 (PIV 1), Parainfluenza virus 2 (PIV 2), Parainfluenza virus 3 (PIV 3), Parainfluenza virus 4 (PIV 4), Human Rhinovirus/Enterovirus (HRV/HEV), Respiratory Syncytial Virus (RSV) A/B, *Bordetella pertussis*, *Chlamydia pneumoniae*, and *Mycoplasma pneumoniae* nucleic acid target sequences. K192485 - Page 1 of 50 {1} # **C Type of Test:** A multiplexed nucleic acid test intended for use with the BioCode MDx-3000 Instrument for the simultaneous qualitative *in vitro* detection and identification of multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swabs (NPS) collected in viral transport media (VTM) or universal transport media (UTM), and obtained from individuals suspected of respiratory tract infections. # **III Intended Use/Indications for Use:** # **A Intended Use(s):** See Indications for Use below. # **B Indication(s) for Use:** The BioCode Respiratory Pathogen Panel (RPP) is a qualitative multiplexed nucleic acid-based *in vitro* diagnostic test intended for use with BioCode MDx-3000 Instrument. The BioCode RPP is capable of the simultaneous detection and identification of nucleic acids from multiple viruses and bacteria extracted from nasopharyngeal swab (NPS) samples obtained from individuals with signs and/or symptoms of respiratory tract infection. The following pathogens and subtypes are identified using the BioCode RPP: - Adenovirus - Coronavirus (229E, OC43, HKU1, and NL63) - Human Metapneumovirus A/B - Influenza A, including subtypes H1, H1 2009 Pandemic, and H3 - Influenza B - Parainfluenza Virus 1 - Parainfluenza Virus 2 - Parainfluenza Virus 3 - Parainfluenza Virus 4 - Respiratory Syncytial Virus A/B - Rhinovirus/Enterovirus - Bordetella pertussis - Chlamydia pneumoniae - Mycoplasma pneumoniae The detection and identification of specific viral and bacterial nucleic acids from individuals exhibiting signs and/or symptoms of a respiratory infection aids in the diagnosis of respiratory infection if used in conjunction with other clinical and epidemiological information. The results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. Negative results in the setting of a respiratory illness may be due to infection with pathogens that are not detected by this test, or lower respiratory tract infection that may not be detected by a nasopharyngeal swab specimen. Positive results do not rule out co-infection with other organisms: the agent(s) detected by the BioCode RPP may not be the definite cause of disease. Additional laboratory testing (e.g., bacterial and viral culture, immunofluorescence, and K192485 - Page 2 of 50 {2} radiography) may be necessary when evaluating a patient with possible respiratory tract infection. Due to the genetic similarity between Human Rhinovirus and Enterovirus, the BioCode RPP cannot differentiate them. A positive BioCode RPP Rhinovirus/Enterovirus result should be followed up using an alternate method (e.g., cell culture or sequence analysis) if differentiation is required. The BioCode RPP detects Human Rhinovirus/Enterovirus with reduced sensitivity. If a more accurate Rhinovirus/Enterovirus result is required, it is recommended that specimens found to be negative for Human Rhinovirus/Enterovirus after examination using BioCode RPP be confirmed by an alternate method (e.g., FDA cleared molecular tests). Performance characteristics for Influenza A were established when Influenza A H1 2009 Pandemic and A H3 were the predominant Influenza A viruses in circulation. Performance of detecting Influenza A may vary if other Influenza A strains are circulating or a novel Influenza A virus emerges. If infection with a novel Influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent Influenza viruses and sent to state or local health departments for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens. # C Special Conditions for Use Statement(s): Rx - For Prescription Use Only # D Special Instrument Requirements: BioCode MDx-3000 Instrument bioMérieux NucliSENS easyMAG system Roche MagNA Pure 96 system # IV Device/System Characteristics: # A Device Description: The BioCode Respiratory Pathogen Panel (RPP) is a respiratory pathogen multiplex nucleic acid test designed for use with the BioCode MDx-3000 system. The BioCode MDx-3000 is an automated system that integrates PCR amplification, target capture, signal generation and optical detection for multiple viral and bacterial pathogens from a single nasopharyngeal swab specimen collected in VTM or UTM. Liquid specimens are processed, and nucleic acids extracted with the bioMérieux NucliSENS easyMAG or Roche MagNA Pure 96 automated systems. Once the PCR plate is manually set up and sealed, all other operations are automated on the MDx-3000. The BioCode RPP simultaneously tests for 17 pathogens and/or subtypes (see the Intended Use section above) from nasopharyngeal swab specimens collected in VTM or UTM. Results from the BioCode RPP test are available within about 5 hours. # Materials Provided with Each BioCode RPP Kit: - BioCode Master Mix A - BioCode RPP Primer Mix - BioCode RT Mix K192485 - Page 3 of 50 {3} - BioCode RNA IC2 - BioCode RPP BMB-Probe Mix - Individually-packaged Transfer Pipettes # Materials Needed but Not Provided with the BioCode RPP Kit: - BioCode SA-PE Mix - BioCode Buffer A10% bleach solution - BioCode MDx-3000 Instrument - BioCode MDx-3000 Consumables: - Reagent Reservoirs (Integra 4332) - Waste Bin and Lid (Applied BioCode 01-W0105) - 20 μL pipette tips (Beckman 717256) - 250 μL pipette tips (Beckman 717252) - 96-well hard shell plate 0.1 mL (Bio-Rad HSL9601) - PCR Adhesive Foil (Thermo Fisher Scientific AB-0626) - Microtiter plate (Greiner bio-one 655101) - Microtiter plate lid (Nunc 5500) - NucliSENS easyMAG (bioMérieux) Extraction System - NucliSENS easyMAG (bioMérieux) Supplies for Extraction: - Lysis Buffer - Buffer 1 - Buffer 2 - Buffer 3 - Magnetic silica - Nuclease free water - Consumables - ELISA strip plate or - MagNA Pure 96 (Roche) Extraction System - MagNA Pure 96 (Roche) Supplies for Extraction: - MagNA Pure 96 DNA and Viral nucleic acid kit - MagNA Pure 96 system fluid - Consumables - Vortex - Centrifuge - Pipettes – single, multi-channel and/or repeater with accuracy range between 1-10 μL, 10-200 μL, and 100 – 1000 μL - Sterile, RNase/DNase-free disposable aerosol-barrier micro pipettor tips - 1.5 mL polypropylene micro centrifuge tubes and racks (RNase/DNase free recommended) - Cooler racks for 1.5 mL tubes and 0.1 mL 96 well plate - Biosafety cabinet (laminar flow hood) for nucleic acids extractions - Freezer (manual defrost) at -10°C to -30°C - Freezer (manual defrost) at -60°C to -90°C - Refrigerator at 2 to 8°C K192485 - Page 4 of 50 {4} ## Interpretation of Results The BioCode MDx-3000 software will analyze data based on plate validity, sample validity and Median Fluorescent Intensity (MFI) compared to an MFI threshold. ## Run Controls and Internal Control Results The BioCode MDx-3000 software will suppress results if Internal or Negative controls are invalid. The software will indicate if external positive controls are valid or invalid but will not suppress results if the positive control is not valid (see *Internal Control*) below. ### *External Negative Controls* External Negative Controls can be RNase-free water, transport media, or well characterized negative specimens. The External Negative Controls should go through all processing steps (extractions, amplification, and detection). At least one External Negative Control is required for each plate/kit lot. The BioCode MDx-3000 software will suppress results for all samples if the External Negative Control is not valid (see Table 1 below). **Table 1: Criteria for Valid External Negative Control** | Control | Targets | RNA IC | Description | | --- | --- | --- | --- | | Negative Control | Not Detected | Detected | Plate Status: Valid. Samples can be interpreted. | | Negative Control | Detected | N/A | Plate Status: Invalid. Samples results cannot be interpreted. Results suppressed by software. | | Negative Control | N/A | Not Detected | Plate Status: Invalid. Samples results cannot be interpreted. Results suppressed by software. | ### *External Positive Controls* External Positive Controls can be well characterized clinical samples or positive strains. External Positive Controls can be single analytes or pooled multianalyte specimens. External Positive Controls should go through all processing steps (extractions, amplification, and detection). It is recommended that at least one External Positive Control be included for each plate/kit lot on a rotating schedule. Wells identified as External Positive Controls will be trended by the BioCode MDx-3000 software and the report will indicate a valid or invalid result on the report header (see Table 2 below). However, the software will not suppress results based on External Positive Control results. If an External Positive Control does not perform as expected, the user should review all samples in that plate to determine if results can be reported. K192485 - Page 5 of 50 {5} Table 2: Criteria for Valid External Positive Control | Control | Targets | RNA IC | Recommendations | | --- | --- | --- | --- | | Positive Control | Expected Target Detected | N/A | Report will indicate Positive Control is Valid. No user intervention required. | | Positive Control | Expected Target Not Detected | N/A | Report will indicate Positive Control is Invalid. User should review results of all samples in that plate prior to release. | | Positive Control | Unexpected Target Detected | N/A | Report will indicate Positive Control is Invalid. User should review results of all samples in that plate prior to release. | # Internal Control An RNA Internal Control (RNA IC: bacteriophage MS2) is added to each sample during extraction. The Internal Control monitors the efficiency of the extraction, reverse transcription, amplification and detection stages of the assay. Positive target assay results may be reported in the absence of RNA IC detection. However, the BioCode MDx-3000 software will suppress target assay negative results for any wells with invalid RNA IC results (see Table 3 below). Lack of RNA IC signal may indicate sample-associated inhibition or reagent/instrumentation issues. Samples suspected of being inhibitory should be repeated from the extraction step. If reagent or instrument issues are suspected specimens may be repeated using residue nucleic acid extracts. Table 3: Criteria for RNA Internal Control (RNA IC) | Targets | RNA IC | Recommendations | | --- | --- | --- | | N/A | Detected | Well status: Valid. Report all results. | | Detected | Not Detected | Well status: Invalid. Detected results may be reported. Consider repeat/reflex testing. | | Not Detected | Not Detected | Well status: Invalid. Not Detected results suppressed by software. Repeat/reflex testing. | # Interpretation of Pathogen Results Fluorescent signals from barcoded magnetic beads (BMBs) with the same barcode are sorted and the median fluorescence index (MFI) is calculated for each analyte assay. The assays are considered “Detected” by comparing the MFI to a specific validated assay cutoff. If assay target specific MFI is at or above the threshold, the assay is positive. For the following organisms detected by the BioCode RPP, the organism is reported as “Detected” if a single corresponding assay is positive. - Rhinovirus/Enterovirus - Parainfluenza Virus 1 - Parainfluenza Virus 2 - Parainfluenza Virus 3 - Parainfluenza Virus 4 - Respiratory Syncytial Virus A/B - Bordetella pertussis K192485 - Page 6 of 50 {6} - Chlamydia pneumoniae - Mycoplasma pneumoniae For the following organisms detected by the BioCode RPP, the organism is reported as “Detected” if at least one of the two corresponding assays is positive. - Influenza B - Human Metapneumovirus A/B - Adenovirus And for the following organism detected by the BioCode RPP, the organism is reported as “Detected” if at least one of the four corresponding assays is positive. - Coronavirus Note: Each of the four Coronavirus (CoV) assays is specifically designed to detect CoV-229E, CoV-OC43, CoV-HKU1, and CoV-NL63, respectively. In addition, the BioCode RPP contains one assay designed to detect Influenza A (FluA), and three hemagglutinin (HA) subtyping assays for A/H1, A/H1pdm09 and A/H3, respectively. Each of the individual assays is interpreted independently and the panel result reported for Influenza A is based on the combined results of the four assays as outlined in Table 4 below. Table 4: Possible Target Assay Results for Influenza A and Corresponding Interpretation | Target Assay BioCode RPP Result | FluA | A/H1 | A/H1pdm09 | A/H3 | Action | | --- | --- | --- | --- | --- | --- | | Influenza A Not Detected | Not Detected | Not Detected | Not Detected | Not Detected | Report result | | Influenza A and A/H1 Detected | Detected | Detected | Not Detected | Not Detected | Report result | | Influenza A and A/H3 Detected | Detected | Not Detected | Not Detected | Detected | Report result | | Influenza A and A/H1pdm09 Detected | Detected | Not Detected | Detected | Not Detected | Report result | | Influenza A/H1, and A/H3 Detected | Detected | Detected | Not Detected | Detected | Multiple infections are possible but rare, retest to confirm result ^{a} | | Influenza A/H1pdm09, and A/H3 Detected | Detected | Not Detected | Detected | Detected | Multiple infections are possible but rare, retest to confirm result ^{a} | | Influenza A/H1, and A/H1pdm09 Detected | Detected | Detected | Detected | Not Detected | Multiple infections are possible but rare, retest to confirm result ^{a} | | Influenza A/H1, A/H1pdm09, and A/H3 Detected | Detected | Detected | Detected | Detected | Multiple infections are possible but rare, retest to confirm result ^{a} | | Influenza A (no subtype detected) | Detected | Not Detected | Not Detected | Not Detected | Retest (see the section on Influenza A, no subtype detected below) | K192485 - Page 7 of 50 {7} | Target Assay BioCode RPP Result | FluA | A/H1 | A/H1pdm09 | A/H3 | Action | | --- | --- | --- | --- | --- | --- | | Influenza A Indeterminate | Not Detected | Detected or Not Detected | Detected or Not Detected | Detected | Retest^{b} | | | Not Detected | Detected | Detected or Not Detected | Detected or Not Detected | | | | Not Detected | Detected or Not Detected | Detected | Detected or Not Detected | | $^{a}$ Repeated multiple positive should be further confirmed by other FDA-cleared influenza subtyping assays. $^{b}$ If the re-test result confirms the original result, it is recommended that the sample be further investigated using a different FDA-cleared influenza A subtyping assay and/or sending the residual sample to local public health laboratory for further testing. ### Influenza A (no subtype detected): If the FluA assay is positive, but none of the hemagglutinin (HA) subtyping assays are positive, then the interpretation is Influenza A (no subtype detected). This result could occur when the titer of the virus in the specimen is low and not detected by the subtyping assays. This result could also indicate the presence of a novel Influenza A strain or a seasonal Influenza A/H3 or A/H1pdm09 strain with critical sequence mismatches to the primers and/or probes of the BioCode RPP influenza A HA subtyping assays. In both cases, the sample in question should be retested. If the retest provides a different result, test the sample a third time to ensure the accuracy of the result. If the retest provides the same result, then the function of the BioCode RPP should be verified by testing with appropriate external control materials (known positive samples for Influenza A/H1, Influenza A/H3 and Influenza A/H1pdm09), and a negative control should also be run to test for PCR-product contamination. If the BioCode RPP accurately identifies the external positive and negative controls, contact the appropriate public health authorities for confirmatory testing. ### BioCode RPP Reports The analyzed BioCode MDx-3000 results are displayed in two report formats: Run Report for the entire run including multiple specimens, and Sample Report for individual specimens. Both reports can be exported as a PDF or CSV file. Each report includes fully analyzed and interpreted results for specimens and/or controls but is formatted differently. The Run Report displays analyzed results in a tabular format for all wells (specimens/controls) in a run from a specific kit lot. If more than one lot is run together, separate Run Reports will be generated by the software for each lot. Possible results by target assay are: Detected, Not detected, Invalid, Indeterminant (for Influenza A only), or N/A (if not ordered). The Sample Report displays results for a single well (specimen/control). In addition to results for each target assay, the Sample Reports include a results summary section which allows positive results to be reviewed at a glance. The Sample Report results summary will also indicate well validity based on BMB counts, background MFI, and external and internal controls. Sample reports also include any sample-specific comments entered during setup. K192485 - Page 8 of 50 {8} Both report headers provide traceability information for: run name, run start and finish time, user ID, software version, instrument ID, kit name, and reagent lots with expiration dates. The report headers also include sections for Run Status and Controls Status. The Run Status section will specify if the run is Incomplete, Valid or Invalid (based on the External Negative Control results for the specific run/kit lot). The Controls Status section indicates the results for the External Negative Controls (Valid or Invalid) and External Positive Controls (Valid, Invalid, or N/A if not assayed). The Run Status and Controls Status sections should be reviewed prior to review of target assay results. In addition, the software will also mask results in the detailed tabular sections based on plate and well validity requirements. Completed reports can be reviewed electronically. Reviewer comments can be added to the report footer for traceability under the review section. In addition, MFI reports are available with information only for administrator level users. # **B Principle of Operation:** The BioCode MDx-3000 is an automated system that integrates PCR amplification, target capture, signal generation, and optical detection for multiple respiratory viruses and bacteria from a single nasopharyngeal swab (NPS) specimen, in either VTM or UTM. Nucleic acids from NPS are extracted with the bioMérieux NucliSENS easyMAG or Roche MagNA Pure 96 automated systems. Once the PCR plate is manually set up and sealed, all other operations are automated on the MDx-3000. # **Nucleic Acid Extraction** Nucleic acids (both RNA and DNA) are captured by coated magnetic beads and eluted on either the bioMérieux NucliSENS easyMAG or the Roche MagNA Pure 96 automated systems according to the respective manufacturer provided protocol. # **Overview of a BioCode MDx-3000 Run:** # **Reverse Transcription and Multiplex PCR** Since targeted pathogens of the BioCode RPP include RNA viruses, a reverse transcription (RT) step is performed to convert the viral RNA into cDNA prior to amplification. The purified nucleic acid solution is manually combined with a freshly prepared reaction mix for the RT step and subsequent thermal cycling for multiplex PCR to enrich the target nucleic acids present in the sample. One of the target-specific primers for each pathogen is biotinylated at the 5'-end to generate labeled PCR product for subsequent detection. # **PCR and Product Transfer** For the PCR amplification, the robotic head dispenses BMB- Probe mix into the designated reaction wells of the capture plate using disposable pipette tips. After PCR amplification is completed, the robotic head pierces the foil seal with disposable pipette tips and transfers PCR products into corresponding wells of the capture plate. # **Target Capture** Amplified PCR products labeled with biotin are captured at a defined temperature by target-specific probes that are covalently coupled to designated Barcoded Magnetic Beads (BMBs). K192485 - Page 9 of 50 {9} During this step, BMBs are kept in suspension by gentle agitation. Differentiation of captured targets is achieved by assigning a unique barcode pattern of BMBs for each pathogen and the Internal Control. ## Signal Generation After washing off unbound PCR products and unused primers, a streptavidin-phycoerythrin (SA-PE) conjugate is automatically added to the reaction by the robot. High affinity binding between biotin and streptavidin ensures that captured PCR products with the biotin moiety are labeled with phycoerythrin in close proximity to the BMBs. ## Optical Detection Optical detection is performed for each reaction well of the capture plate, an optically clear, flat-bottom microtiter plate. After washing off unbound SA-PE, excitation of the fluorophore at the designated wavelength emits fluorescence signal from BMBs tagged with SA-PE conjugates. Each reaction well is imaged at a specific emission wavelength for fluorescent signal and under bright field for identifying the barcode patterns (decoding). ## Interpretation of Results The BioCode MDx-3000 Software controls the operation of the instrument, collects and analyzes data, and automatically generates interpretation for test reports at the end of the run. Fluorescent signals from BMBs with the same barcode are sorted and calculated to generate median fluorescence index (MFI) for each analyte. The presence or absence of a pathogen is determined relative to the validated assay cutoff by MFI. The software also analyzes the results of external and internal controls to validate the run and individual specimen results for reporting. C Instrument Description Information: | Modes of Operation | Yes | No | | --- | --- | --- | | Does the applicant's device contain the ability to transmit data to a computer, webserver, or mobile device? | ☑ | ☐ | | Does the applicant's device transmit data to a computer, webserver, or mobile device using wireless transmission? | ☐ | ☑ | | Software | | | | FDA has reviewed applicant's Hazard Analysis and software development processes for this line of product types. | ☑ | ☐ | 1. Instrument Name: BioCode MDx-3000 Instrument 2. Specimen Identification: Specimen identity is provided by barcode magnetic beads. 3. Specimen Sampling and Handling: After extraction with the NucliSENS easyMag system or the MagNA Pure 96 system and manually loading samples into a 96-well formatted plate, the BioCode MDx 3000 processes all RT-PCR, target capture, signal generation, and optical detection steps automatically. K192485 - Page 10 of 50 {10} # 4. Calibration: Optical calibration of the BioCode MDx 3000 is performed twice a year by Applied BioCode. No calibration kit is available. # 5. Quality Control: Each laboratory should establish its own Quality Control ranges and frequency of QC testing based on applicable local laws, regulations, and good laboratory practices. The BioCode RPP uses an internal control (bacteriophage MS2) which is added to each sample prior to extraction. The internal control monitors the efficiency of the extraction, reverse transcription, amplification, and detection stages of the assay. Positive results may be reported in the absence of RNA IC detection. The BioCode RPP software will suppress negative results for any wells with invalid RNA IC results. # V Substantial Equivalence Information: # A Predicate Device Name(s): FilmArray Respiratory Panel 2 (RP2) # B Predicate 510(k) Number(s): K170604 # C Comparison with Predicate(s): | Similarities or Differences | | | | --- | --- | --- | | Element | BioCode Respiratory Pathogen Panel (RPP) (K192485) | FilmArray Respiratory Panel 2 (RP2) (K170604) | | Specimen Types | NPS (in VTM or UTM) | Same | | Pathogens Detected | Adenovirus, Coronavirus (229E, HKU1, NL63, and OC43), Human Metapneumovirus A/B, Influenza A, Influenza A subtype H1, Influenza A subtype H3, Influenza A subtype 2009 Pandemic, Influenza B, Parainfluenza Virus 1, Parainfluenza Virus 2, Parainfluenza Virus 3, Parainfluenza Virus 4, Rhinovirus/Enterovirus, Respiratory Syncytial Virus A/B, *Bordetella pertussis*, *Chlamydia pneumoniae*, and *Mycoplasma pneumoniae* | Same, except that the four coronaviruses, 229E, HKU1, NL63, and OC43, are detected and reported separately, and the pathogens detected also include *Bordetella parapertussis* | | Analyte | RNA/DNA | Same | | Technological Principles | Multiplex nucleic acid | Same | | Instrumentation | BioCode MDx-3000 Instrument, bioMérieux NucliSENS easyMAG system or Roche MagNA Pure 96 system | FilmArray 2.0 or FilmArray Torch | | Time to Result | About 5.0 hours | About 45 minutes | | Test Interpretation | Automated test interpretation and report generation. | Same | K192485 - Page 11 of 50 {11} | Controls | An RNA Internal Control is added to each sample during extraction. External Positive and Negative Controls are externally sourced. | Two controls are included in each reagent pouch to control for sample processing and both stages of PCR and melt analysis. External Positive and Negative Controls are externally sourced. | | --- | --- | --- | | Methodology | Multiplex RT-PCR in a single reaction and probe hybridization followed by fluorescence detection and decoding of barcoded magnetic beads (BMB) that capture biotinylated PCR products with streptavidin conjugate. | Nested multiplex PCR executed in two stages. First, a single, large volume, highly multiplexed reverse transcription PCR (RT-PCR) reaction. Second, nested PCR, is performed in singleplex fashion in each well of the array, followed by melting curve analyses of the PCR reactions. | | CLIA Complexity | High | Moderate | ## VI Standards/Guidance Documents Referenced: - FDA guidance document issued on August 27, 2014, titled “Highly Multiplexed Microbiological/Medical Countermeasure In Vitro Nucleic Acid Based Diagnostic Devices” - FDA guidance document issued on October 9, 2009, titled “Class II Special Controls Guidance Document: Respiratory Viral Panel Multiplex Nucleic Acid Assay” - FDA guidance document issued on October 9, 2009, titled “Class II Special Controls Guidance Document: Testing for Detection and Differentiation of Influenza A Virus Subtypes Using Multiplex Assays” - FDA guidance document issued on October 9, 2009, titled “Testing for Human Metapneumovirus (hMPV) Using Nucleic Acid Assays” - FDA guidance document issued on July 15, 2011, titled “Establishing the Performance Characteristics of In Vitro Diagnostic Devices for the Detection or Detection and Differentiation of Influenza Viruses” - FDA guidance document issued on April 25, 2005, titled “Guidance on Informed Consent for In Vitro Diagnostic Device Studies Using Leftover Human Specimens that are Not Individually Identifiable” ## VII Performance Characteristics (if/when applicable): ### A Analytical Performance: #### 1. Precision/Reproducibility: A reproducibility study was conducted at three testing sites using a combination of NucliSENS easyMAG and MagNA Pure 96 automated systems for nucleic acids extraction (two sites used NucliSENS easyMAG and one site used MagNA Pure 96 for nucleic acids extraction). The study incorporated potential sources of variation introduced by site (three testing sites), day (five different days), and operator (two operators per site). One lot of reagents was assayed at three sites by two operators on one BioCode MDx-3000 instrument per site for five days. A total of 10 BioCode RPP runs were performed per site. A total of six contrived NPS samples containing known quantities of various BioCode RPP analytes (Table 5 below) were prepared in a simulated NPS in UTM sample matrix. A NPS sample negative for all BioCode RPP analytes was also prepared using the simulated NPS in UTM sample matrix. The six contrived positive samples consisted of combinations of 12 K192485 - Page 12 of 50 {12} representative pathogens, at 1.5x LoD (Low) and 3x LoD (Medium), spiked into a simulated NPS in UTM sample matrix. Table 5: Reproducibility Study Panel – Sample Composition | Medium (3x LoD) | Medium (3x LoD) | Low (1.5x LoD) | Low (1.5x LoD) | Sample Name | | --- | --- | --- | --- | --- | | Rhinovirus | Parainfluenza Virus 2 | Human Metapneumovirus | Bordetella pertussis | RP1 | | Human Metapneumovirus | Bordetella pertussis | Rhinovirus | Parainfluenza Virus 2 | RP2 | | Influenza B | Coronavirus NL63 | Chlamydia pneumoniae | Parainfluenza Virus 3 | RP3 | | Chlamydia pneumoniae | Parainfluenza Virus 3 | Influenza B | Coronavirus NL63 | RP4 | | Influenza A H3N2 | Mycoplasma pneumoniae | Respiratory Syncytial Virus | Adenovirus C | RP5 | | Respiratory Syncytial Virus | Adenovirus C | Influenza A H3N2 | Mycoplasma pneumoniae | RP6 | | Simulated Negative NPS in UTM matrix | | | | RP7 | Information regarding the specific strains and isolates of the 12 representative pathogens and concentrations that were used in building the reproducibility study panel samples are provided in Table 6 below. Table 6: Reproducibility Study Panel - Strains/Isolates and Concentrations | Pathogen | Strain/Serotype | Source/ ID | Limit of Detection (LoD) | Low Concentration (1.5xLoD) | Medium Concentration (3xLoD) | | --- | --- | --- | --- | --- | --- | | Coronavirus NL63 | NL63 | Zeptometrix 0810228CF | 0.04 TCID_{50}/mL | 0.06 TCID_{50}/mL | 0.12 TCID_{50}/mL | | Parainfluenza virus 2 | Greer/Ohio/1955 | ATCC VR-92 | 5.4 TCID_{50}/mL | 8.1 TCID_{50}/mL | 16.2 TCID_{50}/mL | | Adenovirus C | Species C | ATCC VR-846 | 18.0 TCID_{50}/mL | 27.0 TCID_{50}/mL | 54.0 TCID_{50}/mL | | Influenza A H3N2 | A/Wisconsin/67/05 | Zeptometrix 0810252CF | 4.0 TCID_{50}/mL | 6.0 TCID_{50}/mL | 12.0 TCID_{50}/mL | | Human Rhinovirus | Type 1A | Zeptometrix 0810012CF | 1.2 TCID_{50}/mL | 1.8 TCID_{50}/mL | 3.6 TCID_{50}/mL | | Mycoplasma pneumoniae | M129 | Zeptometrix 0801579 | 15.0 CCU/mL | 22.5 CCU/mL | 45.0 CCU/mL | | Chlamydia pneumoniae | AR39 | ATCC VR-53592 | 33.3 CFU/mL | 50.0 CFU/mL | 100.0 CFU/mL | | Influenza B | Florida/4/2006 (Yamagata) | Zeptometrix 0810255CF | 0.01 TCID_{50}/mL | 0.02 TCID_{50}/mL | 0.03 TCID_{50}/mL | | Parainfluenza virus 3 | N/A | Zeptometrix 0810060CF | 15.0 TCID_{50}/mL | 22.5 TCID_{50}/mL | 45.0 TCID_{50}/mL | | Human Metapneumovirus | Type 16, Type A1 IA10-2003 | Zeptometrix 0810161CF | 15.0 TCID_{50}/mL | 22.5 TCID_{50}/mL | 45.0 TCID_{50}/mL | | Respiratory Syncytial Virus | Type A | Zeptometrix 0810040ACF | 0.33 TCID_{50}/mL | 0.45 TCID_{50}/mL | 0.9 TCID_{50}/mL | | Bordetella pertussis | A639 | Zeptometrix 0801459 | 45.0 CFU/mL | 67.5 CFU/mL | 135.0 CFU/mL | Once prepared, each sample of the reproducibility study panel was tested with the BioCode RPP to confirm that it contained the intended analytes at the intended concentration and then divided into single-use aliquots and stored frozen (≤ -70°C) until the day of testing. K192485 - Page 13 of 50 {13} After being distributed to the sites, each sample was extracted in triplicates using either the NucliSENS easyMAG (Site 1 and Site 3) or the MagNA Pure 96 (Site 2) and assayed using the BioCode RPP. In total, 90 data points per sample were obtained, with 60 data points generated by the NucliSENS easyMAG system (Site 1 and Site 3) and 30 data points generated by the MagNA Pure 96 system. A summary of the reproducibility study results, percent (%) agreement with the expected Detected or Not Detected result, for each analyte (by site and extraction system) is provided in Table 7 below. Table 7: Reproducibility of BioCode RPP | Analyte | Concentration Tested | Expected Result | Agreement with Expected Result | | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | | NucliSENS easyMAG | | | MagNA Pure 96 | | All Sites (95% CI) | | | | | Site 1 | Site 3 | Sub-Total | Site 2 | Sub-Total | | | **Viruses** | | | | | | | | | | Adenovirus | 3× LoD | Detected | 30/30 100% | 30/30 100% | 60/60 100% | 30/30 100% | 30/30 100% | 90/90 100% (95.9%-100%) | | | 1.5× LoD | Detected | 30/30 100% | 30/30 100% | 60/60 100% | 30/30 100% | 30/30 100% | 90/90 100% (95.9%-100%) | | | None (no analyte) | Not Detected | 150/150 100% | 149/150 99.3% | 299/300 99.7% | 150/150 100% | 150/150 100% | 449/450 99.8% (98.8%-100%) | | Coronavirus | 3× LoD | Detected | 30/30 100% | 30/30 100% | 60/60 100% | 30/30 100% | 30/30 100% | 90/90 100% (95.9%-100%) | | | 1.5× LoD | Detected | 30/30 100% | 30/30 100% | 60/60 100% | 30/30 100% | 30/30 100% | 90/90 100% (95.9%-100%) | | | None (no analyte) | Not Detected | 150/150 100% | 150/150 100% | 300/300 100% | 150/150 100% | 150/150 100% | 450/450 100% (99.2%-100%) | | Human Metapneumovirus | 3× LoD | Detected | 30/30 100% | 30/30 100% | 60/60 100% | 30/30 100% | 30/30 100% | 90/90 100% (95.9%-100%) | | | 1.5× LoD | Detected | 30/30 100% | 30/30 100% | 60/60 100% | 30/30 100% | 30/30 100% | 90/90 100% (95.9%-100%) | | | None (no analyte) | Not Detected | 150/150 100% | 150/150 100% | 300/300 100% | 150/150 100% | 150/150 100% | 450/450 100% (99.2%-100%) | | Human Rhinovirus/Enterovirus | 3× LoD | Detected | 30/30 100% | 30/30 100% | 60/60 100% | 30/30 100% | 30/30 100% | 90/90 100% (95.9%-100%) | | | 1.5× LoD | Detected | 30/30 100% | 30/30 100% | 60/60 100% | 30/30 100% | 30/30 100% | 90/90 100% (95.9%-100%) | | | None (no analyte) | Not Detected | 150/150 100% | 150/150 100% | 300/300 100% | 150/150 100% | 150/150 100% | 450/450 100% (99.2%-100%) | K192485 - Page 14 of 50 {14} | Analyte | Concentration Tested | Expected Result | Agreement with Expected Result | | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | | NucliSENS easyMAG | | | MagNA Pure 96 | | All Sites (95% CI) | | | | | Site 1 | Site 3 | Sub-Total | Site 2 | Sub-Total | | | Influenza A/H3 | 3× LoD | Detected | 30/30 100% | 30/30 100% | 60/60 100% | 30/30 100% | 30/30 100% | 90/90 100% (95.9%-100%) | | | 1.5× LoD | Detected | 29/30^{a} 96.7% | 30/30 100% | 59/60 98.3% | 30/30 100% | 30/30 100% | 89/90 98.9% (94.0%-99.8%) | | | None (no analyte) | Not Detected | 150/150 100% | 149/150^{b} 99.3% | 299/300 99.7% | 150/150 100% | 150/150 100% | 449/450 99.8% (98.8%-100%) | | Influenza A/H1pdm09 | None (no analyte) | Not Detected | 210/210 100% | 210/210 100% | 420/420 100% | 210/210 100% | 210/210 100% | 630/630 100% (99.4%-100%) | | Influenza A/H1 | None (no analyte) | Not Detected | 210/210 100% | 210/210 100% | 420/420 100% | 210/210 100% | 210/210 100% | 630/630 100% (99.4%-100%) | | Influenza B | 3× LoD | Detected | 30/30 100% | 30/30 100% | 60/60 100% | 30/30 100% | 30/30 100% | 90/90 100% (95.9%-100%) | | | 1.5× LoD | Detected | 29/30 96.7% | 30/30 100% | 59/60 98.3% | 30/30 100% | 30/30 100% | 89/90 98.9% (94.0%-99.8%) | | | None (no analyte) | Not Detected | 150/150 100% | 150/150 100% | 300/300 100% | 150/150 100% | 150/150 100% | 450/450 100% (99.2%-100%) | | Parainfluenza Virus 1 | None (no analyte) | Not Detected | 210/210 100% | 210/210 100% | 420/420 100% | 210/210 100% | 210/210 100% | 630/630 100% (99.4%-100%) | | Parainfluenza Virus 2 | 3× LoD | Detected | 30/30 100% | 30/30 100% | 60/60 100% | 30/30 100% | 30/30 100% | 90/90 100% (95.9%-100%) | | | 1.5× LoD | Detected | 30/30 100% | 30/30 100% | 60/60 100% | 30/30 100% | 30/30 100% | 90/90 100% (95.9%-100%) | | | None (no analyte) | Not Detected | 150/150 100% | 150/150 100% | 300/300 100% | 150/150 100% | 150/150 100% | 450/450 100% (99.2%-100%) | K192485 - Page 15 of 50 {15} | Analyte | Concentration Tested | Expected Result | Agreement with Expected Result | | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | | NucliSENS easyMAG | | | MagNA Pure 96 | | All Sites (95% CI) | | | | | Site 1 | Site 3 | Sub-Total | Site 2 | Sub-Total | | | **Parainfluenza Virus 3** | **3× LoD** | **Detected** | **30/30** 100% | **30/30** 100% | **60/60** 100% | **30/30** 100% | **30/30** 100% | **90/90** **100%** (95.9%-100%) | | | **1.5× LoD** | **Detected** | **30/30** 100% | **30/30** 100% | **60/60** 100% | **30/30** 100% | **30/30** 100% | **90/90** **100%** (95.9%-100%) | | | **None** (no analyte) | **Not Detected** | **150/150** 100% | **150/150** 100% | **300/300** 100% | **150/150** 100% | **150/150** 100% | **450/450** **100%** (99.2%-100%) | | **Parainfluenza Virus 4** | **None** (no analyte) | **Not Detected** | **210/210** 100% | **210/210** 100% | **420/420** 100% | **210/210** 100% | **210/210** 100% | **630/630** **100%** (99.4%-100%) | | **Respiratory Syncytial Virus** | **3× LoD** | **Detected** | **29/30** 100% | **30/30** 100% | **59/60** 98.3% | **30/30** 100% | **30/30** 100% | **89/90** **98.9%** (94.0%-99.8%) | | | **1.5× LoD** | **Detected** | **29/30** 100% | **30/30** 100% | **59/60** 98.3% | **30/30** 100% | **30/30** 100% | **89/90** **98.9%** (94.0%-99.8%) | | | **None** (no analyte) | **Not Detected** | **150/150** 100% | **150/150** 100% | **300/300** 100% | **150/150** 100% | **150/150** 100% | **450/450** **100%** (99.2%-100%) | | **Bacteria** | | | | | | | | | | *Mycoplasma pneumoniae* | **3× LoD** | **Detected** | **30/30** 100% | **30/30** 100% | **60/60** 100% | **30/30** 100% | **30/30** 100% | **90/90** **100%** (95.9%-100%) | | | **1.5× LoD** | **Detected** | **30/30** 100% | **30/30** 100% | **60/60** 100% | **30/30** 100% | **30/30** 100% | **90/90** **100%** (95.9%-100%) | | | **None** (no analyte) | **Not Detected** | **150/150** 100% | **149/150** 99.3% | **299/300** 99.7% | **150/150** 100% | **150/150** 100% | **449/450** **99.8%** (98.8%-100%) | | *Bordetella pertussis* | **3× LoD** | **Detected** | **30/30** 100% | **30/30** 100% | **60/60** 100% | **30/30** 100% | **30/30** 100% | **90/90** **100%** (95.9%-100%) | | | **1.5× LoD** | **Detected** | **30/30** 100% | **30/30** 100% | **60/60** 100% | **30/30** 100% | **30/30** 100% | **90/90** **100%** (95.9%-100%) | | | **None** (no analyte) | **Not Detected** | **150/150** 100% | **150/150** 100% | **300/300** 100% | **150/150** 100% | **150/150** 100% | **450/450** **100%** (99.2%-100%) | | *Chlamydia pneumoniae* | **3× LoD** | **Detected** | **30/30** 100% | **30/30** 100% | **60/60** 100% | **30/30** 100% | **30/30** 100% | **90/90** **100%** (95.9%-100%) | | | **1.5× LoD** | **Detected** | **30/30** 100% | **30/30** 100% | **60/60** 100% | **30/30** 100% | **30/30** 100% | **90/90** **100%** (95.9%-100%) | | | **None** (no analyte) | **Not Detected** | **150/150** 100% | **150/150** 100% | **300/300** 100% | **150/150** 100% | **150/150** 100% | **450/450** **100%** (99.2%-100%) | *There was an indeterminate Flu A result for Influenza A/H3 low positive sample. K192485 - Page 16 of 50 {16} b There was an indeterminate Flu A result for Influenza A/H3 negative sample. 2. Linearity: Not applicable, qualitative assay 3. Assay Reportable Range: Not applicable, qualitative assay 4. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods): # Assay Controls # External Negative Controls External Negative Controls are user provided. They can be RNase-free water, transport media, or well characterized negative specimens. The External Negative Controls should go through all processing steps (extractions, amplification, and detection). At least one External Negative Control is required for each plate/kit lot. The BioCode MDx-3000 software will suppress results for all samples if the External Negative Control is not valid. # External Positive Controls External Positive Controls are user provided. They can be well characterized clinical samples or cultured organisms. External Positive Controls can be single analytes or pooled multianalyte specimens. External Positive Controls should go through all processing steps (extractions, amplification, and detection). It is recommended that at least one External Positive Control be included for each plate/kit lot on a rotating schedule. Wells identified as External Positive Controls will be trended by the BioCode MDx-3000 software and the report will indicate a valid or invalid result on the report header. However, the software will not suppress results based on External Positive Control results. If an External Positive Control does not perform as expected, the user should review all samples in that plate to determine if results can be reported. # Internal Control An RNA Internal Control (RNA IC: bacteriophage MS2) is provided in the reagent kit and is added to each sample during extraction. The Internal Control monitors the efficiency of the extraction, reverse transcription, amplification, and detection stages of the assay. Positive target assay results may be reported in the absence of RNA IC detection. However, the BioCode MDx-3000 software will suppress target assay negative results for any wells with invalid RNA IC results. External controls are not provided with the BioCode RPP. However, six frozen (-70°C) external control mixes obtained from ZeptoMetrix as NATtrol controls for different pathogens (see Table 8 below) were provided to the clinical study sites for testing during the prospective clinical study and the clinical study testing retrospective and contrived specimens. Operators were required to follow the BioCode RPP instructions for use during the testing. K192485 - Page 17 of 50 {17} Table 8: External Control Mixes Utilized in the Clinical Evaluations | External Control Mixes | Expected Calls | | --- | --- | | PC-A | Influenza B, Respiratory Syncytial Virus, Parainfluenza Virus 1, and Rhinovirus/Enterovirus | | PC-B | Coronavirus HKU1, Parainfluenza Virus 2, Human Metapneumovirus, and Bordetella pertussis | | PC-C | Influenza A/H1, Coronavirus NL63, and Adenovirus | | PC-D | Influenza A/H1pdm09, Parainfluenza Virus 4, Coronavirus OC43, and Chlamydia pneumoniae | | PC-E | Influenza A/H3, Parainfluenza Virus 3, Coronavirus 229E, and Mycoplasma pneumoniae. | | NC | Negative for all analytes | Performance of testing the external controls during the clinical studies are summarized in Table 9 below. Table 9: External Controls Testing Summary | | Agreement with Expected Results | | | | | | | --- | --- | --- | --- | --- | --- | --- | | | PC-A | PC-B | PC-C | PC-D | PC-E | NC | | NucliSENS easyMAG | 9/10^{a} | 8/9^{a} | 6/6 | 11/11 | 8/8 | 50/53^{b} | | MagNA Pure 96 | 4/4 | 5/5 | 4/4 | 3/3 | 3/3 | 20/20 | | Total | 13/14 | 13/14 | 10/10 | 14/14 | 11/11 | 70/73 | $^{a}$PC and NC switched during loading due to operator's error. $^{b}$Of the three failed NCs, one was due to RNA IC failure, one due to operator's error (NC and PC switched during loading), and one due to detection of an unexpected target. # Specimen Stability Study Nasopharyngeal Swab (NPS) should be collected according to standard technique and immediately placed in 1 – 3 mL of VTM or UTM. Samples should be tested as soon as possible. An analytical study was performed to assess the specimen stability limitations for the optimal performance of the BioCode RPP on the BioCode MDx-3000. Nine representative pathogens from the BioCode RPP were spiked into prescreened negative natural NPS in UTM specimens at 3x LoD (see Table 10 below) and assayed with three extraction replicates on the easyMAG at each timepoint, except for baseline. A total of seven extraction replicates on the easyMAG were assayed at timepoint 0. Stability of extracted nucleic acids was also assessed in this study. Table 10: Contrived Sample in Prescreened Negative Natural NPS in UTM Utilized in the Specimen Stability Study | Sample Name | Pathogen Name and Concentration | | --- | --- | | RP A | Adenovirus (ADV at 3xLoD), Mycoplasma pneumoniae (MPN at 3xLoD), and Influenza A/H3 (Flu A/H3 at 3xLoD) | | RP B | Respiratory Syncytial Virus (RSV at 3xLoD), Influenza A/H1pdm09 (Flu A/H1pdm09 at 3xLoD), and Human Metapneumovirus (hMPV at 3xLoD) | | RP C | Parainfluenza Virus 3 (PIV 3 at 3xLoD), Coronavirus NL63 (CoV-NL63 at 3xLoD), and Influenza B (Flu B at 3xLoD) | Specimen stability study summary results are presented in Table 11 below. K192485 - Page 18 of 50 {18} Table 11: Specimen Stability Study Summary Results | Sample Type | Temperature | Time (hours or Days) | Agreement with Expected Results | | | | | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | | Sample RP A | | | Sample RP B | | | Sample RP C | | | | | | | ADV | MPN | Flu A/H3 | RSV | Flu A/H1 pdm09 | hMPV | PIV 3 | CoV-NL63 | Flu B | | Fresh (baseline) | N/A | 0 | 7/7 | 7/7 | 7/7 | 7/7 | 7/7 | 7/7 | 7/7 | 7/7 | 7/7 | | Contrived NPS | Room Temp | 8 Hour | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | | | | 12 Hour | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | | | 4°C | 5 Day | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | | | | 7 Day | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | | | | 10 Day | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | | | -80°C | 30 Day | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | | | | 60 Day | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | | | | 90 Day | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | | Extracted Nucleic Acids | 4°C | 8 Hour | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | | | | 12 Hour | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | | | -80°C (2x Freeze/Thaw) | 30 Day | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | | | | 60 Day | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | | | | 90 Day | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | The specimen stability testing data supports the following specimen stability claims in the product package insert: NPS collected in VTM or UTM may be stored at room temperature for 8 hours, in a 2-8°C refrigerator for 7 days, or at <-60°C for up to 90 days. Extracted nucleic acids may be stored in a 2-8°C refrigerator for up to 12 hours, or at ≤-60°C for up to 90 days. ### Fresh vs. Frozen The results of the Specimen Stability Study (see Table 11 above) also demonstrated that preservation of samples (by freezing at -80°C) does not affect the accuracy of test results compared to freshly prepared samples. Therefore, it is appropriate to utilize frozen archived prospective and retrospective clinical samples in the evaluation of BioCode RPP to supplement the prospective (fresh) clinical study data, and to use frozen simulated samples in analytical studies for this submission. ### Single-Spiked vs. Multi-Spiked Specimen Study An analytical study was conducted to assess the performance of the BioCode RPP on the BioCode MDx-3000 with mixed analyte samples at or near the Limit of Detection (LoD) compared to single spiked samples to justify the use of multiple analyte spiked samples during analytical validation testing. LoD was determined for single spiked samples in simulated NPS for four representative pathogens on both the easyMAG and MagNA pure extraction systems. For initial range finding, four replicates of each concentration were extracted on the easyMAG and MagNA pure systems and tested in singlet with the BioCode RPP on the BioCode MDx-3000 system to estimate LoD. The LoD was further confirmed by extracting 20 replicates of each sample and testing each in singlet for a total of 20 replicates at or near the presumptive LoD. The single spiked LoDs were then challenged by pooling all K192485 - Page 19 of 50 {19} four pathogens and testing at 1x LoD with 20 extraction replicates on both extraction systems. When 1x LoD for each pathogen in the mixed pool does not meet the 95% detection goal, the pooled pathogen samples were retested at 3x LoD with 20 extraction replicates. Results within 3x LoD were considered to be equivalent in this study. Summary results of this study are presented in Table 12 below. Table 12: Single-Spiked vs. Multi-Spiked Study Summary Results | Pathogen | Target Assay | Single-Spiked or Multi-Spiked | NucliSENS easyMAG | | MagNA Pure 96 | | | --- | --- | --- | --- | --- | --- | --- | | | | | Concentration | Detection (n/20) | Concentration | Detection (n/20) | | Influenza A/H3N2 | Flu A | Single-Spiked | 1.3 TCID_{50}/mL | 20/20 | 1.3 TCID_{50}/mL | 20/20 | | | Flu A/H3 | | | 20/20 | | 20/20 | | | Flu A | Multi-Spiked | 4.0 TCID_{50}/mL | 20/20 | | 20/20 | | | Flu A/H3 | | | 20/20 | | 20/20 | | Coronavirus NL63 | NL 63 | Single-Spiked | 0.013 TCID_{50}/mL | 20/20 | 0.040 TCID_{50}/mL | 19/20 | | | | Multi-Spiked | 0.040 TCID_{50}/mL | 20/20 | | 20/20 | | *Mycoplasma pneumoniae* | MPN | Single-Spiked | 5.0 CCU/mL | 19/20 | 15.0 CCU/mL | 20/20 | | | | Multi-Spiked | 15.0 CCU/mL | 20/20 | | 20/20 | | Adenovirus C | ADV1 | Single-Spiked | 6.0 TCID_{50}/mL | 20/20 | 18.0 TCID_{50}/mL | 20/20 | | | | Multi-Spiked | | 20/20 | | 20/20 | The acceptance criteria of this study were met since all pathogens tested were within 3xLoD for single-spiked and multi-spiked samples. The study results seem to demonstrate that the performance of the BioCode RPP on the BioCode MDx-3000 with mixed analyte samples at or near the Limit of Detection (LoD) is similar to single spiked samples. Using multiple analyte spiked samples during analytical validation testing appears to be acceptable. ### Simulated vs. Natural NPS in UTM Specimen Study Analytical validation studies are mostly performed with contrived specimens (i.e., spiking known concentrations of pathogens into negative NPS in UTM or VTM matrix). This would require a large volume of negative natural NPS to be collected and screened, while typically only between 1.0 to 2.0 mL per donor could be obtained after NPS collection and screening to confirm negative status. This makes collection from donors burdensome. An analytical study was performed to assess the performance equivalency of testing a simulated NPS (sNPS) in UTM matrix compared to testing natural NPS in UTM using the BioCode RPP, with both the NucliSENS easyMAG and the MagNA Pure 96 extraction systems. The sNPS in UTM matrix consists of 2x10³ HeLa cells/mL diluted in UTM. Samples were contrived in negative natural NPS in UTM matrix and in sNPS in UTM matrix with multi-spiked pathogens at close to LoD levels (approximately 1.5x to 15.0xLoD). Each sample was extracted in quadruplicate on both the easyMAG and the MagNA Pure 96 extraction system and tested singly with the BioCode RPP on the BioCode MDx-3000 system. Summary results of this analytical study are presented in Table 13 below. K192485 - Page 20 of 50 {20} Table 13: Simulated vs. Natural NPS in UTM Specimen Study Summary Results | Pools | Pathogen | Concentration | Detected (N of 4) | | | | | --- | --- | --- | --- | --- | --- | --- | | | | | NucliSENS easyMAG | | MagNA Pure 96 | | | | | | Natural NPS in UTM | sNPS in UTM | Natural NPS in UTM | sNPS in UTM | | A | Adenovirus E Serotype 4 | 0.6 TCID50/mL (15xLoD) | 4/4 | 4/4 | 4/4 | 4/4 | | | Chlamydia pneumoniae | 75.0 CFU/mL (2.3 - 4.5xLoD) | 4/4 | 4/4 | 4/4 | 4/4 | | | Influenza A/H3N2 | 6.0 TCID50/mL (1.5 -4.6xLoD) | 4/4 | 4/4 | 4/4 | 4/4 | | B | Influenza A/H1N1 | 67.5 TCID50/mL (4.5 - 13.5xLoD) | 4/4 | 4/4 | 4/4 | 4/4 | | | Respiratory Syncytial Virus A | 1.5 TCID50/mL (4.5xLoD) | 4/4 | 4/4 | 4/4 | 4/4 | | | Human Metapneumovirus | 67.5 TCID50/mL (4.5xLoD) | 4/4 | 4/4 | 4/4 | 4/4 | | C | Influenza B | 0.06 TCID50/mL (6.0xLoD) | 4/4 | 4/4 | 4/4 | 4/4 | | | Coronavirus OC43 | 0.06 TCID50/mL (1.5 - 6.0xLoD) | 4/4 | 4/4 | 4/4 | 4/4 | | | Parainfluenza Virus 4 | 13.5 TCID50/mL (1.5xLoD) | 4/4 | 4/4 | 4/4 | 4/4 | | Negative Control | | N/A | 4/4 | 4/4 | 4/4 | 4/4 | The study results show that the performance testing contrived samples in this simulated NPS (sNPS) in UTM matrix compared to testing contrived samples in natural NPS in UTM matrix using the BioCode RPP, with both the NucliSENS easyMAG and the MagNA Pure 96 extraction systems, are similar. Therefore, it is acceptable to utilize the simulated NPS in UTM sample matrix in conducting the analytical studies in support of this submission. ### 5. Detection Limit: Limit of detection (LoD) estimation and confirmation studies were carried out with contrived samples in simulated NPS in UTM (Remel M4 transport medium) matrix designed to resemble a natural clinical NPS in UTM specimen. An equivalence study was performed which demonstrated that the simulated matrix was similar to the natural clinical NPS matrix and did not impact BioCode RPP test performance. Refer to the “Simulated vs. Natural NPS in UTM Specimen Study” section. Representative isolates of respiratory viruses and bacteria were selected to make contrived samples in order to obtain positive results for every target assay on the panel. In some cases, testing of more than one isolate/strain per analyte was performed to assess LoD for clinically important species or variants, specifically when more than one assay was needed to detect the expected diversity of an analyte (e.g. Adenovirus). An estimated LoD concentration for each analyte was first determined by testing serial dilutions of contrived samples. Four extraction replicates per dilution on each extraction system, NucliSENS easyMAG and MagNA Pure 96, were tested using the BioCode RPP. The LoD was confirmed by extracting 20 replicates on each extraction system and testing each for a total of 20 replicates at or near the estimated LoD using the BioCode RPP. LoD for each isolate was defined as the lowest concentration with \( \geq95\% \) detection of 20 replicates (at least 19 out of 20 replicates). A multi-spiked approach was employed in both the LoD estimation and the LoD confirmation studies where samples spiked with two pathogens at various concentrations were tested in the LoD studies. An equivalence study was performed which demonstrated that the performance of the multi-spiked samples was similar to that of the single-spiked K192485 - Page 21 of 50 {21} samples and employing a multi-spiking approach did not impact BioCode RPP test performance. Refer to the “Single-Spiked vs. Multi-Spiked Specimen Study” section. Summary results of the LoD confirmation study are presented in Table 14 below. Table 14: Limit of Detection Confirmation by Extraction System | Pathogen | Strain/Isolate | Source | NucliSENS easyMAG | | MagNA Pure 96 | | | --- | --- | --- | --- | --- | --- | --- | | | | | Concentration | Detected (n of 20) | Concentration | Detected (n of 20) | | Influenza A/H1 | A/New/Caledonia/20/99 | Zeptometrix 0810036CF | 15.0 TCID_{50}/mL | 20/20 | 5.0 TCID_{50}/mL | 20/20 | | | A/NWS/33 | ATCC VR-219 | 27.0 TCID_{50}/mL | 20/20 | 9.0 TCID_{50}/mL | 20/20 | | Influenza A/H1pdm09 | A(H1N1)/California/07/09 | Zeptometrix 0810165CF | 0.4 TCID_{50}/mL | 20/20 | 0.4 TCID_{50}/mL | 20/20 | | Influenza A/H3 | A/Wisconsin/67/05 | Zeptometrix 0810252CF | 4.0 TCID_{50}/mL | 20/20 | 1.3 TCID_{50}/mL | 20/20 | | | A/Alice | ATCC VR-776 | 27.0 TCID_{50}/mL | 20/20 | 9.0 TCID_{50}/mL | 19/20 | | Influenza B | B/Florida/4/2006 (Yamagata) | Zeptometrix 0810255CF | 0.01 TCID_{50}/mL | 20/20 | 0.01 TCID_{50}/mL | 20/20 | | | B/Hong Kong/S/1972 (Victoria) | ATCC VR-823 | 48.6 TCID_{50}/mL | 20/20 | 48.6 TCID_{50}/mL | 20/20 | | Respiratory Syncytial Virus | Type A | Zeptometrix 0810040ACF | 0.33 TCID_{50}/mL | 20/20 | 0.33 TCID_{50}/mL | 20/20 | | Human Metapneumovirus | 16; Type A1 IA10-2003 | Zeptometrix 0810161CF | 15.0 TCID_{50}/mL | 19/20 | 15.0 TCID_{50}/mL | 20/20 | | Parainfluenza Virus 1 | C-35/Washington DC/1957 | ATCC VR-94 | 9.0 TCID_{50}/mL | 20/20 | 9.0 TCID_{50}/mL | 20/20 | | Parainfluenza Virus 2 | Geer/Ohio/1955 | ATCC VR-92 | 1.8 TCID_{50}/mL | 20/20 | 5.4 TCID_{50}/mL | 20/20 | | Parainfluenza Virus 3 | N/A | Zeptometrix 0810016CF | 15.0 TCID_{50}/mL | 20/20 | 15.0 TCID_{50}/mL | 20/20 | | Parainfluenza Virus 4 | Type 4a | Zeptometrix 0810060CF | 9.0 TCID_{50}/mL | 20/20 | 9.0 TCID_{50}/mL | 20/20 | | Adenovirus | Species B Serotype 7A | Zeptometrix 0810021CF | 1.2 TCID_{50}/mL | 20/20 | 1.2 TCID_{50}/mL | 20/20 | | | Species C Serotype 2 | ATCC VR-846 | 6.0 TCID_{50}/mL | 20/20 | 18.0 TCID_{50}/mL | 20/20 | | | Species E Serotype 4 | Zeptometrix 0810070CF | 0.04 TCID_{50}/mL | 20/20 | 0.04 TCID_{50}/mL | 19/20 | | Coronavirus 229E | N/A | Zeptometrix 0810229CF | 0.6 TCID_{50}/mL | 20/20 | 1.8 TCID_{50}/mL | 20/20 | | Coronavirus HKU1 | N/A | Clinical Sample 4922^{a} | 5.02x10^{4} Copies/mL | 19/20 | 5.02x10^{4} Copies/mL | 20/20 | | Coronavirus NL63 | N/A | Zeptometrix 0810228CF | 0.04 TCID_{50}/mL | 20/20 | 0.04 TCID_{50}/mL | 20/20 | | Coronavirus OC43 | N/A | Zeptometrix 0810024CF | 0.04 TCID_{50}/mL | 20/20 | 0.01 TCID_{50}/mL | 19/20 | | Rhinovirus | Type A1 | Zeptometrix 0810012CF | 1.2 TCID_{50}/mL | 20/20 | 0.4 TCID_{50}/mL | 19/20 | | Enterovirus | D68 | Zeptometrix 0810300CF | 3.0 TCID_{50}/mL | 19/20 | 9.0 TCID_{50}/mL | 20/20 | | Bordetella pertussis | A639 | Zeptometrix 801459 | 15.0 CFU/mL | 20/20 | 45.0 CFU/mL | 19/20 | | Chlamydia pneumoniae | AR39 | ATCC VR-53592 | 16.7 CFU/mL | 20/20 | 33.3 CFU/mL | 20/20 | | Mycoplasma pneumoniae | M129 | Zeptometrix 0801579 | 15.0 CCU/mL | 20/20 | 15.0 CCU/mL | 20/20 | $^{a}$ Coronavirus HKU1 clinical sample quantified with Applied BioCode validated SYBR assay using an IVT RNA standard. K192485 - Page 22 of 50 {22} ## 6. Analytical Reactivity: An analytical study was performed to assess analytical reactivity/inclusivity of the BioCode RPP. Different strains, serotypes and genotypes were selected that represent temporal, geographic, and genetic diversity for each pathogen. A panel of titered stocks for relevant organisms/viruses diluted in simulated NPS in UTM matrix were tested starting at 3x LoD. Organisms/viruses not detected at 3x LoD were tested at higher concentrations. Since the confirmed LoD result for B/Hong Kong/S/1972 (Victoria lineage) was higher than expected at 48.6 TCID \( _{50} \) /mL, which could be related to difference in titration from vendor sources, testing of the influenza B Victoria strains in this study was performed starting at 0.03xLoD or 0.3xLoD. Strains of unknown lineages of influenza B were tested at 3x LoD for the B/Florida/4/2006 strain of the Yamagata lineage. Each sample was extracted in triplicate on the NucliSENS easyMAG and tested with the BioCode RPP on the BioCode MDx-3000 according to the instructions for use. Note: Although this analytical reactivity testing was performed in part to demonstrate that the BioCode RPP can detect various species and strains of a pathogen with similar analytical sensitivity, it is difficult to make comparative sensitivity evaluations between different organisms and different strains that have been quantified in \( TCID_{50}/mL \) or \( EID_{50}/mL \) . This quantification method measures the infectivity and cytotoxicity or lethality of a strain in tissue culture or in chick embryo, and is therefore subject to many influences (e.g., strain-to-strain differences in infectivity, viability of the original material, culturing conditions, etc.). Quantification by infectivity assay will not be equivalent between strains or organisms. Also, since the measurand for the BioCode RPP is nucleic acids, LoD concentrations established in \( TCID_{50}/mL \) or \( EID_{50}/mL \) can vary dramatically among different strains but may not actually reflect significant differences in analytical reactivity of detection as measured by target nucleic acids concentration. Analytical reactivity testing results are summarized in Table 15 to Table 25 below. Table 15: Adenovirus Isolates Tested and Detected by BioCode RPP | Species\( ^{a} \) | Serotype | Isolate ID/Source | Strain/Location/Year | xLoD Detected | Result | | --- | --- | --- | --- | --- | --- | | A | 31 | Zeptometrix 0810073CF | Unknown | 3x | Adenovirus Detected | | | 12 | ATCC VR-863 | Huie/Massachusetts | 3x | | | | 18 | ATCC VR-19 | Washington DC/1954 | 3x | | | B | 3 | ATCC VR-3 | GB/Maryland/1953 | 3x | | | | 14 | Zeptometrix 0810108CF | Unknown | 3x | | | | 16 | ATCC VR-17 | CH.79/Saudi Arabia/1955 | 3x | | | | 35 | ATCC VR-718 | Holden | 3x | | | C | 1 | Zeptometrix 0810050CF | Unknown | 3x | | | | 5 | Zeptometrix 0810020CF | Unknown | 3x | | | | 6 | ATCC VR-6 | Tonsil 99/Washington DC | 3x | | | D | 8 | Zeptometrix 0810069CF | Unknown | 3x | | | | 17 | ATCC VR-1836 | CH.22/Saudi Arabia/1955 | 3x | | | | 20 | Zeptometrix 0810115CF | Unknown | 3x | | | | 26 | Zeptometrix 0810117CF | Unknown | 3x | | | | 37 | Zeptometrix 0810119CF | Unknown | 3x | | | F | 40 | Zeptometrix 0810084CF | Unknown | 3x | | | | 41 | ATCC VR-930 | Tak/73-3544/Netherlands/1973 | 3x | | \( ^{a} \) In addition to the Adenovirus species E serotype 4 strain tested in the LoD study, in silico analysis of available sequences predicts that the BioCode RPP will also react with other Adenovirus species E serotypes. K192485 - Page 23 of 50 {23} Table 16: Coronavirus Isolates/Specimens Tested and Detected by BioCode RPP | Coronavirus Type | Isolate ID/Source | Location/Year | xLoD Detected | Result | | --- | --- | --- | --- | --- | | 229E | ATCC VR-740 | Unknown | 3x | Coronavirus Detected | | NL63 | BEI NR-470a | Amsterdam/2003 | 3x | Coronavirus Detected | | OC43 | ATCC VR-1558 | Unknown | 3x | Coronavirus Detected | | HKU1 | Clinical Sample 5016 | Unknown | 3x | Coronavirus Detected | | | Clinical Sample 5036 | Unknown | 3x | | | | Clinical Sample 5037 | Unknown | 10x | | \( ^{a} \) Organism obtained through BEI Resources, NIAID, NIH: Human Coronavirus NL63, NR-470. Table 17: Human Metapneumovirus Isolates Tested and Detected by BioCode RPP | Genotype | Serotype | Isolate ID/Source | Location/Year | xLoD Detected | Result | | --- | --- | --- | --- | --- | --- | | A1 | 9 | Zeptometrix 0810160CF | Iowa3/2002 | 3x | Human Metapneumovirus Detected | | A2 | 27 | Zeptometrix 0810164CF | Iowa27/2004 | 3x | | | B1 | 3 | Zeptometrix 0810156CF | Peru2/2002 | 3x | | | | 5 | Zeptometrix 0810158CF | Peru3/2003 | 3x | | | B2 | 4 | Zeptometrix 0810157CF | Peru1/2002 | 3x | | | | 8 | Zeptometrix 0810159CF | Peru6/2003 | 3x | | | | 18 | Zeptometrix 0810162CF | Iowa18/2003 | 3x | | | | Unknown | BEI NR-22232a | TN/91-316 | 3x | | \( ^{a} \) Virus obtained through BEI Resources, NIAID, NIH: Human Metapneumovirus, TN/91-316, NR-22232. Table 18: Human Rhinovirus and Enterovirus Isolates Tested and Detected by BioCode RPP | Species | Serotype/Strain/Isolate | Source | xLoD Detected | Result | | --- | --- | --- | --- | --- | | Human Rhinovirus | | | | | | A | Serotype 7 [68-CV11] | ATCC VR-1601 | 3x | Human Rhinovirus/Enterovirus Detected | | | Serotype 16 [1A] | Zeptometrix 0810285CF | 3x | | | | Serotype 16[11757/Washington DC/1960] | ATCC VR-283 | 3x | | | | Serotype 34 [173-3] | ATCC VR-1365 | 3x | | | | Serotype 57 [ch47] | ATCC VR-1600 | 3x | | | | Serotype 77 [130-63] | ATCC VR-1187 | 3x | | | | Serotype 80 | Zeptometrix 0810288CF | 3x | | | | Serotype 85 [50-525-CV54] | ATCC VR-1195 | 3x | | | | Serotype 95 [SF-998] | ATCC VR-1301 | 3x | | | B | Serotype 3 [FEB] | ATCC VR-483 | 3x | | | | Serotype 14 | Zeptometrix 0810284CF | 3x | | | | Serotype 42 | Zeptometrix 0810286CF | 3x | | | | Serotype 70 | Zeptometrix 0810287CF | 3x | | | Enterovirus | | | | | | A | Enterovirus 71 | Zeptometrix 0810236CF | 3x | Human Rhinovirus/Enterovirus Detected | K192485 - Page 24 of 50 {24} Table 19: Influenza A Isolates Tested and Detected by BioCode RPP | Influenza A Typea | Strain | Source | xLoD Detected | Result | | --- | --- | --- | --- | --- | | Influenza A H1N1 | Solomon Island/03/06 | Zeptometrix 0810036CFN | 3x | Influenza A/H1 Detected | | | Singapore/63/04 | Zeptometrix 0810246CF | 3x | | | | PR/8/34 | Zeptometrix 0810245CF | 3x | | | | A/Brisbane/59/2007 | Zeptometrix 0810244CF | 3x | | | | A/Taiwan/42/06 | Zeptometrix 0810247CF | 3x | | | | A/New jersey/8/76 | ATCC VR-897 | \( 500x^b \) | | | | A/Denver/1/1957 | VIRAPUR | 3x | | | | A/FM/1/47 | ATCC VR-97 | 3x | | | | A/Weiss/43 | ATCC VR-96 | \( 1000x^c \) | | | | A/Beijing/262/95d | BEI NR-12277 | \( 30x^e \) | | | | A/Mal/302/54 | ATCC VR-98 | 3x | | | Influenza A H1N2 | Recombinant; Kilbourne F64, A/NWS/1934 (HA) x A/Rockefeller Institute/ 5/1957 (NA)f | BEI NR-3682 | \( 5x^g \) | | | Influenza A H1N1 pdm09 | NY/01/09 | Zeptometrix 0810248CF | 3x | Influenza A/H1 pdm09 Detected | | | NY/02/09 | Zeptometrix 0810109CFN | 3x | | | | NY/03/09 | Zeptometrix 0810249CF | 3x | | | | A/Houston/3H/2009 (H1N1) pdm09b | BEI NR-20340 | 3x | | | | Influenza A H1N1pdm09 (Canada/6294/09) | Zeptometrix 0810109CFJ | 3x | | | | Influenza A H1N1pdm09 (Mexico/4108/09) | Zeptometrix 0810166CF | 3x | | | | California/04/09, cell isolatei | BEI NR-13658 | \( 100x^j \) | | | | A/Christ Church/16/2010 | CDC 2010839805 | \( 1000x^k \) | | | | A/Brisbane/02/2018 | CDC 3000683658 | \( 100x^l \) | | | Influenza A H3N2 | A/Wisconsin/15/2009m | BEI NR-42007 | 3x | Influenza A/H3 Detected | | | A/Texas/50/12 | Zeptometrix 0810238CF | 3x | | K192485 - Page 25 of 50 {25} | | A/Brisbane/10/2007 | Zeptometrix 0810138CF | 3x | | | --- | --- | --- | --- | --- | | | A/Port Chalmers/1/73 | ATCC VR-810 | 50x^{h} | | | | A/Victoria/3/1975 | VIRAPUR | 3x | | | | A/Victoria/361/2011^{o} | BEI NR-44022 | 3x | | | | A/Victoria/3/75 | ATCC VR-822 | 3x | | | | A/Uruguay/716/07^{p} | BEI NR-42003 | 10x | | | | A/HK/H090-756-V1(0)/2009^{q} | BEI NR-44344 | 3x | | | | A/Hong Kong/8/68 | Zeptometrix 0810250CF | 3x | | | | A/Switzerland/9715293/13 | VIRAPUR | 3x | | | | A/Aichi/2/68 | ATCC VR-547 | 3x | | | | MRC-2 | ATCC VR-777 | 3x | | | | A/Perth/16/2009 | CDC 2009719818 | 10x | | | | A/Kansas/14/2017 | CDC 3026015402 | 2000x^{r} | | $^{a}$ In silico analysis supports detection of Influenza A H2N3, H5N1, H5N2, H5N3, H5N8, H7N7, H7N9, H3N1, H3N2, H3N5, H3N7, H3N8 as Influenza A. However, predicted reactivities of the subtyping assays for these influenza A strains of animal origin are variable. $^{b}$ Detected as Flu A (no subtype) at 3x LoD. Detected as dual positive A/H1 and A/H1pdm09 at 500x LoD. $^{c}$ Detected as Flu A (no subtype) at 100x LoD. In silico analysis showed several mismatches in the forward primer for the Flu A/H1 subtyping assay which may account for the observed lower sensitivity of the Flu A/H1 subtyping assay for this strain. $^{d}$ Virus obtained through BEI Resources, NIAID, NIH: Influenza A, A/Beijing/262/95 (H1N1), NR-12277. $^{e}$ Detected as Flu A (no subtype) at 10xLoD. In silico analysis showed a G-A mismatch in the 3' terminal position in the forward primer for the Flu A/H1 subtyping assay which may account for the observed lower sensitivity of the Flu A/H1 subtyping assay for this strain. $^{f}$ Recombinant Virus obtained through BEI Resources, NIAID, NIH: Influenza A, Kilbourne F64, A/NWS/1934 (HA) x A/Rockefeller Institute/5/1957 (NA)] (H1N2), NR-3682 $^{g}$ Detected as Flu A (no subtype) at 3x LoD. $^{h}$ Virus obtained through BEI Resources, NIAID, NIH: Influenza A, A/Houston/3H/2009 (H1N1) pdm09, NR-20340 $^{i}$ Virus obtained through BEI Resources, NIAID, NIH: Influenza A, A/California/04/09, cell isolate (H1N1) pdm09, NR-13658 $^{j}$ In silico analysis of a partial sequence of this strain for the Flu A/H1pdm09 subtyping assay does not predict reduced analytical reactivity. Titering inconsistency from the vendor rather than reduced reactivity due to assay design is suggested. $^{k}$ Virus obtained through the CDC Influenza Division. In silico analysis of partial sequences of this strain for the Flu A/H1pdm09 HA subtyping assay and the Flu A matrix gene assay does not predict reduced analytical reactivity. Titering inconsistency from the source laboratory (EID$_{50}$/mL vs. TCID$_{50}$/mL) rather than reduced reactivity due to assay design is suggested. $^{l}$ Virus obtained through the CDC Influenza Division. In silico analysis did not reveal any critical mismatch in the Flu A/H1pdm09 HA subtyping assay primers and probe binding regions or any mismatch in the FluA matrix gene assay primers and probe binding regions that would predict reduced analytical reactivity. Titering inconsistency from the source laboratory (EID$_{50}$/mL vs. TCID$_{50}$/mL) rather than reduced reactivity due to assay design is suggested. $^{m}$ Virus obtained through BEI Resources, NIAID, NIH: Influenza A, A/Wisconsin/15/2009 (H3N2), NR-42007 K192485 - Page 26 of 50 {26} \( ^{a} \) In silico analysis revealed a few non-critical mismatches in the FluA/H3 subtyping HA assay probe binding region that could contribute to the observed lower reactivity. However, titering inconsistency from the vendor rather than reduced reactivity due to assay design is suspected. \( ^{a} \) Virus obtained through BEI Resources, NIAID, NIH: Influenza A, A/Victoria/361/2011 (H3N2), NR-44022 \( ^{p} \) Virus obtained through BEI Resources, NIAID, NIH: Influenza A, A/Uruguay/716/07 (H3N2), NR-42003 \( ^{9} \) Virus obtained through BEI Resources, NIAID, NIH: Influenza A, A/HK/H090-756-V1(0)/2009 (H3N2), NR-44344 \( ^{1} \) Virus obtained through the CDC Influenza Division. This strain was detected by BioCode RPP as Flu A (no subtype) at 50xLoD. In silico analysis did not reveal any mismatch in the Flu A matrix assay primers and probe binding regions but showed 3 mismatches in the Flu A/H3 subtyping HA assay primers binding regions that could predict reduced analytical reactivity, 1 mismatch at the 9 \( ^{th} \) position from the 3' end of the reverse primer (mismatch #1), 1 mismatch at the 18 \( ^{th} \) position from the 3' end of the reverse primer (mismatch #2), and 1 mismatch at the 20 \( ^{th} \) position from the 3' end of the reverse primer (mismatch #3). Wet testing data suggested that mismatch #1 is likely the root cause for the observed significant reduction in analytical reactivity for this strain. Based on an in silico analysis, of all the Flu A/H3 strains isolated in 2019 with published HA sequences, 73.8% of the strains harbor mismatch #1. Therefore, for patient samples contain a Flu A/H3 strain that harbors mismatch #1 at lower concentrations, the BioCode RPP will likely report a Flu A (no subtype detected) result. Table 20: Influenza B Isolates Tested and Detected by BioCode RPP | Lineage | Strain | Source | xLoD Detected | Result | | --- | --- | --- | --- | --- | | Unknown | B/Lee/1940 | Zeptometrix 0810257CF | 3x | Influenza B Detected | | | B/Taiwan/2/1962 | ATCC VR-295 | \( 500x^a \) | | | | B/Allen/45 | ATCC VR-102 | 5x | | | | B/Brigit/Russia/1969 | ATCC VR-786 | 3x | | | Victoriab | B/Malaysia/2506/2004c | BEI NR-9723 | 0.03x | | | | B/Malaysia/2506/2004 | Zeptometrix 0810258CF | 0.03x | | | | B/Ohio/01/2005d | BEI NR-41801 | 0.3x | | | | B/Brisbane/33/2008e | BEI NR-42006 | 0.3x | | | | B/Nevada/03/2011f | BEI NR-44023 | 0.03x | | | | B/Michigan/09/2011 | CDC 2012700901 | 0.3x | | | | B/Colorado/06/2017 | CDC 3025629447 | 0.3x | | | Yamagata | B/Texas/06/2011g | BEI NR-44024 | \( 5000x^h \) | | | | B/Sydney/507/2006i | BEI NR-36526 | \( 800x^j \) | | | | B/Wisconsin/1/10 | Zeptometrix 0810241CF | 3x | | | | B/Massachusetts/2/12 | Zeptometrix 0810239CF | 3x | | | | B/Christchurch/33/2004k | BEI NR-36536 | 3x | | | | B/New Hampshire/01/2016l | CDC 3000415764 | \( 1000x^l \) | | | | B/Phuket/3073/2013m | CDC 2014768616 | \( 500x^m \) | | K192485 - Page 27 of 50 {27} \( ^{a} \) In silico analysis could not be performed due to unavailability of sequence information for this strain. Titering inconsistency from the vendor rather than reduced reactivity due to assay design is suspected. \( ^{b} \) For Victoria strains testing started at 0.03x LoD rather than 3x LoD. \( ^{c} \) Virus obtained through BEI Resources, NIAID, NIH: Influenza B, B/Malaysia/2506/2004, NR-9723 \( ^{d} \) Virus obtained through BEI Resources, NIAID, NIH: Influenza B, B/Ohio/01/2005, NR-41801 \( ^{e} \) Virus obtained through BEI Resources, NIAID, NIH: Influenza B, B/Brisbane/33/2008, NR-42006 \( ^{f} \) Virus obtained through BEI Resources, NIAID, NIH: Influenza B, B/Nevada/03/2011, NR-44023 \( ^{g} \) Virus obtained through BEI Resources, NIAID, NIH: Influenza B, B/Texas/06/2011, NR-44024 \( ^{h} \) In silico analysis does not predict reduced analytical reactivity. Titering inconsistency from the vendor rather than reduced reactivity due to assay design is suggested. \( ^{i} \) Virus obtained through BEI Resources, NIAID, NIH: Influenza B, B/Sydney/507/2006, NR-36526 \( ^{j} \) In silico analysis does not predict reduced analytical reactivity. Titering inconsistency from the vendor rather than reduced reactivity due to assay design is suggested. \( ^{k} \) Virus obtained through BEI Resources, NIAID, NIH: Influenza B, B/Christchurch/33/2004, NR-36536 \( ^{1} \) Virus obtained through the CDC Influenza Division. In silico analysis did not reveal any critical mismatch in the Flu B NS1 assay primers and probe binding regions that would predict reduced analytical reactivity. And In silico analysis did not reveal any mismatch in the Flu B matrix assay primers and probe binding regions. Titering inconsistency from the source laboratory (EID \( _{50} \) /mL vs. TCID \( _{50} \) /mL) rather than reduced reactivity due to assay design is suggested. \( ^{m} \) Virus obtained through the CDC Influenza Division. In silico analysis did not reveal any critical mismatch in the Flu B NS1 assay primers and probe binding regions that would predict reduced analytical reactivity. And In silico analysis did not reveal any mismatch in the Flu B matrix assay primers and probe binding regions. Titering inconsistency from the source laboratory (EID \( _{50} \) /mL vs. TCID \( _{50} \) /mL) rather than reduced reactivity due to assay design is suggested. Table 21: Parainfluenza Virus Isolates Tested and Detected by BioCode RPP | Type | Subtype | Strain | Source | xLoD Detected | Result | | --- | --- | --- | --- | --- | --- | | 1 | FRA/27344044/2007a | BEI NR-48681 | 3x | Parainfluenza Virus 1 Detected | | | | | FRA/29221106/2009b | BEI NR-48680 | | 3x | | | | Unknown | Zeptometrix 0810014CF | | 3x | | 2 | Greerc | BEI NR-3229 | 3x | Parainfluenza Virus 2 Detected | | | | | Unknown | Zeptometrix 0810015CF | | 3x | | 3 | NIH 47885, Wash/47885/57d | BEI NR-3233 | 3x | Parainfluenza Virus 3 Detected | | | | | C-243/Washington DC/1957 | ATCC VR-93 | | 3x | | 4 | a | M-25/1958e | BEI NR-3237 | 3x | Parainfluenza Virus 4 Detected | | | b | CH-19503/Washington DC/1962 | ATCC VR-1377 | 3x | | | | | Unknown | Zeptometrix 0810060BCF | 3x | | \( ^{a} \) Virus obtained through BEI Resources, NIAID, NIH: Parainfluenza Virus 1, HIPIV1/FRA/27344044/2007, NR-48681 \( ^{b} \) Virus obtained through BEI Resources, NIAID, NIH: Parainfluenza Virus 1, HPIV1/FRA/29221106/2009, NR-48680 \( ^{c} \) Virus obtained through BEI Resources, NIAID, NIH: Parainfluenza Virus 2, Greer, NR-3229 \( ^{d} \) Virus obtained through BEI Resources, NIAID, NIH: Parainfluenza Virus 3, NIH 47885, NR-3233 \( ^{e} \) Virus obtained through BEI Resources, NIAID, NIH: Parainfluenza Virus 4a, M-25, NR-3237 K192485 - Page 28 of 50 {28} Table 22: Respiratory Syncytial Virus Isolates Tested and Detected by BioCode RPP | Type | Strain | Source | xLoD Detected | Result | | --- | --- | --- | --- | --- | | A | TN/1998/3-2^{a} | BEI NR-28529 | 3x | Respiratory Syncytial Virus Detected | | | TN/2000/3-4^{b} | BEI NR-28530 | 10x | | | | TN/98/12-21^{c} | BEI NR-28528 | 3x | | | | Long/Maryland/1956 | ATCC VR-26 | 10x | | | B | 9320/Massachusetts/1977 | ATCC VR-955 | 3x | | | | B1^{d} | BEI NR-4052 | 10x | | | | WV/14617/1985 | ATCC VR-1400 | 3x | | | | 18537/Washington DC/1962 | ATCC VR-1580 | 3x | | | | CH-93 (18)-18 | Zeptometrix 0810040CF | 3x | | $^{a}$ Virus obtained through BEI Resources, NIAID, NIH: RSV, TN/1998/3-2, NR-28529 $^{b}$ Virus obtained through BEI Resources, NIAID, NIH: RSV, TN/2000/3-4, NR-28530 $^{c}$ Virus obtained through BEI Resources, NIAID, NIH: RSV, TN/98/12-21, NR-28528 $^{d}$ Virus obtained through BEI Resources, NIAID, NIH: RSV, B1, NR-4052 Table 23: Bordetella pertussis Isolates Tested and Detected by BioCode RPP | Isolate | Source | xLoD Detected | Result | | --- | --- | --- | --- | | F | ATCC 8467 | 3x | Bordetella pertussis Detected | | 5[17921] | ATCC 9340 | 3x | | | 10-536 | ATCC 10380 | 3x | | | CNCTC Hp 12/63,623 | ATCC 51445 | 3x | | | Tohama 1 | ATCC BAA-589 | 3x | | | MN2531 | ATCC BAA-1335 | 3x | | Table 24: Chlamydia pneumoniae Isolates Tested and Detected by BioCode RPP | Isolate | Source | xLoD Detected | Result | | --- | --- | --- | --- | | CM-1/Georgia | ATCC VR-1360 | 3x | Chlamydia pneumoniae Detected | | CWL-029 | ATCC VR-1310 | 3x | | Table 25: Mycoplasma pneumoniae Isolates Tested and Detected by BioCode RPP | Isolate | Source | xLoD Detected | Result | | --- | --- | --- | --- | | M129-B7 | ATCC 29342 | 3x | Mycoplasma p…
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