(53 days)
The Diagnostic Hybrids, Inc. device, D3 DFA Metapneumovirus Identification Kit, is intended for the qualitative detection and identification of human metapneumovirus (hMPV) in nasal and nasopharyngeal swabs and aspirates/washes or cell culture. The assay detects hMPV antigens by immunofluorescence using a blend of three monoclonal antibodies (MAbs), from patients with signs and symptoms of acute respiratory infection. This assay detects but is not intended to differentiate the four recognized genetic sub-lineages of hMPV.
Negative results do not preclude hMPV infection and should not be used as the sole basis for diagnosis, treatment or other management decisions. It is recommended that specimens found to be negative after examination of the direct specimen results be confirmed by an FDA cleared hMPV molecular assay.
The D DFA Metapneumovirus Identification Kit uses a blend of three hMPV antigen-specific murine MAbs that are directly labeled with fluorescein for detection of hMPV. The reagent detects but does not differentiate between the four recognized subtypes of hMPV (subtypes A1, A2, B1, and B2).
Kit Components:
-
- Metapneumovirus DFA Reagent, 5-mL. One dropper bottle containing a blend (see below for MAb discussion) of fluorescein-labeled murine monoclonal antibodies directed against MPV. The buffered, stabilized, aqueous solution contains Evans Blue as a counter-stain and 0.1% sodium azide as a preservative.
-
- hMPV Antigen Control Slides, 5 slides. Five individually packaged control slides, each with a well containing cell culture-derived MPV positive cells and a well containing cell culture-derived negative cells. Each slide is intended to be stained only one time. Control material has been treated to be non-infectious; however normal laboratory precautions are required when the material is handled.
-
- 40X PBS Concentrate, 25-mL. One bottle containing a 40X concentrate consisting of 4% sodium azide (0.1% sodium azide after dilution to 1X using de-mineralized water) in PBS.
-
- Mounting Fluid, 7-mL. One dropper bottle containing an aqueous, buffer-stabilized solution of glycerol and 0.1% sodium azide.
The cells to be tested, derived from a clinical specimen or cell culture, are placed onto a glass slide, allowed to air dry and are fixed in acetone. The Metapneumovirus DFA Reagent is added to the cells which are then incubated for 15 to 30 minutes at 35℃ to 37℃ in a humidified chamber or humidified incubator. The stained cclls are then washed with the diluted phosphate buffered saline (PBS), a drop of the supplied Mounting Fluid is added and a coverslip is placed on the prepared cells. The cells are examined using a fluorescence microscope. The hMPV infected cells will fluoresce apple-green. Uninfected cells will contain no fluorescence but will be stained red by the Evans Blue counter-stain.
It is recommended that specimens found to contain no fluorescent cells after examination of the direct specimen be confirmed by an FDA cleared hMPV molecular assay.
Here's an analysis of the acceptance criteria and the study that proves the device meets them, based on the provided text:
Device Name: D3 DFA Metapneumovirus Identification Kit
1. Table of Acceptance Criteria and Reported Device Performance
The acceptance criteria are implied by the performance results required for clearance. As this is not a modern AI/ML device, the performance metrics are clinical sensitivity and specificity, or Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA). Acceptance usually implies that these metrics meet certain thresholds, particularly with lower bounds of the 95% Confidence Interval (CI) demonstrating adequate performance.
For Direct Specimen Testing (Clinical Studies Sites 1-3):
| Metric | Acceptance Criteria (Implied) | Reported Device Performance (Site 1: Nasal Wash/Aspirate) | Reported Device Performance (Site 2: Nasal/Nasopharyngeal Swab) | Reported Device Performance (Site 3: Nasal Wash/Aspirate)* | Reported Device Performance (Site 3: Nasal/Nasopharyngeal Swab)* |
|---|---|---|---|---|---|
| Sensitivity | Sufficiently high (e.g., >80% or >90%, with acceptable CI) | 53.0% (95% CI: 46.6%-59.5%) | 70.7% (95% CI: 57.3%-81.9%) | PPA: 100.0% (95% CI: 66.4%-100%) | PPA: 75.0% (95% CI: 19.4%-99.4%) |
| Specificity | Sufficiently high (e.g., >90% or >95%, with acceptable CI) | 99.8% (95% CI: 99.3%-99.9%) | 99.7% (95% CI: 98.2%-100%) | NPA: 100.0% (95% CI: 85.2%-100%) | NPA: 100.0% (95% CI: 94.2%-100%) |
*Note: For Study Site 3, Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) were reported instead of sensitivity and specificity due to the comparator method used.
For Cultured Cells Testing (Clinical Study Site 4):
| Metric | Acceptance Criteria (Implied) | Reported Device Performance (Site 4: Freeze-thawed Nasopharyngeal Swab Amplified in Cell Culture)* |
|---|---|---|
| Sensitivity | Sufficiently high | PPA: 83.3% (95% CI: 35.9%-99.6%) |
| Specificity | Sufficiently high | NPA: 100.0% (95% CI: 99.7%-100%) |
For Reproducibility (Analytical Performance):
| Metric | Acceptance Criteria (Implied) | Reported Device Performance |
|---|---|---|
| Agreement with Expected Result (Positive) | 100% agreement | 100% (120/120) |
| Agreement with Expected Result (Negative) | 100% agreement | 100% (90/90) |
| Total Percent Agreement | 100% agreement | 100% (210/210) |
2. Sample Size Used for the Test Set and Data Provenance
The clinical performance studies used the following sample sizes for the test set:
- Study Site 1: 1482 fresh nasal wash/nasopharyngeal aspirate specimens.
- Study Site 2: 368 fresh nasal/nasopharyngeal swab specimens.
- Study Site 3: 32 fresh nasal wash/nasopharyngeal aspirate specimens and 66 fresh nasal/nasopharyngeal swab specimens.
- Study Site 4 (Cultured Cells): 74 freeze-thawed nasopharyngeal swab specimens that were cultured.
Data Provenance: The data was collected during prospective studies at 3 geographically diverse U.S. clinical laboratories (Study Sites 1-3) during the 2005-2006 and 2006-2007 respiratory virus seasons (December 2005 - April 2006 and December 2006 - March 2007). Study Site 4, for cultured cells, was performed at DHI during the 2007-2008 respiratory virus season (January - April 2008). Specimens were "excess, remnants of respiratory specimens that were prospectively collected from symptomatic individuals suspected of respiratory infection, and were submitted for routine care or analysis by each site, and that otherwise would have been discarded." Individual specimens were de-linked from all patient identifiers. All clinical sites were granted waivers of informed consent by their IRBs.
3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications of Those Experts
The document does not explicitly state the number of experts or their qualifications for establishing the ground truth. It describes the ground truth as composite comparator methods or a single comparator assay in different sites:
- Sites 1 and 2: Ground truth was a composite comparator method consisting of viral culture and a validated real-time RT-PCR comparator assay with bi-directional sequencing analysis. "True" hMPV positive was defined as positive by viral culture OR positive by RT-PCR with sequencing matching hMPV. "True" hMPV negative was defined as negative by both viral culture and RT-PCR.
- Site 3 and 4: Ground truth was based solely on the validated hMPV real-time RT-PCR followed by bi-directional sequencing analysis comparator assay. Positive was defined by sequencing data matching hMPV, and negative by negative RT-PCR.
While these methods are considered reference standards in microbiology, the document does not specify human expert involvement in interpreting these results or adjudicating discrepancies, beyond the inherent expertise in running and interpreting these laboratory assays.
4. Adjudication Method for the Test Set
The document does not explicitly detail an "adjudication method" in the sense of multiple human readers resolving disagreements, as would be typical for image-based AI studies. Instead, the ground truth itself is a carefully defined reference standard.
- For Sites 1 and 2, the ground truth was a composite definition:
- Positive: Viral culture positive OR Real-time RT-PCR positive with bi-directional sequencing matching hMPV.
- Negative: Viral culture negative AND Real-time RT-PCR negative.
- For Sites 3 and 4, the ground truth was based on the hMPV real-time RT-PCR followed by bi-directional sequencing analysis comparator assay. Positive was defined by acceptable sequencing data matching hMPV, and negative by negative RT-PCR.
This structure inherently handles potential discrepancies between methods by prioritizing certain outcomes (e.g., a positive by either method for the composite ground truth).
5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done, If So, What Was the Effect Size of How Much Human Readers Improve with AI vs. Without AI Assistance
No, an MRMC comparative effectiveness study involving human readers and AI assistance was not done. This device is a diagnostic kit (an immunofluorescence assay), not an AI-powered system designed to assist human readers in interpreting complex data. The clinical studies evaluated the standalone performance of the kit itself against a reference standard.
6. If a Standalone (i.e. algorithm only, without human-in-the-loop performance) Was Done
Yes, the studies described are essentially standalone performance studies for the D3 DFA Metapneumovirus Identification Kit. The kit's results (fluorescence detection by microscopy) were compared directly against the established ground truth without any involvement of a human-in-the-loop for interpreting the kit's results in the context of an AI system. The "algorithm" here is the biochemical and optical detection mechanism of the DFA test combined with human interpretation of fluorescence under a microscope, as per standard laboratory practice.
7. The Type of Ground Truth Used
The type of ground truth used varied slightly across study sites but primarily involved molecular and classical microbiological methods:
- Clinical Study Sites 1 and 2: Composite Ground Truth combining:
- Viral Culture: A classical microbiological method for isolating and identifying viruses.
- Validated Real-time RT-PCR followed by bi-directional sequencing analysis: A molecular method to detect hMPV nucleic acid, with sequencing confirming the identity.
- Clinical Study Sites 3 and 4: Molecular Ground Truth based solely on a validated hMPV real-time RT-PCR followed by bi-directional sequencing analysis. This method is considered a highly specific and sensitive reference standard.
8. The Sample Size for the Training Set
No information about a "training set" for an algorithm is provided. This device is a diagnostic kit (DFA assay), not an AI/ML model that undergoes a training phase.
9. How the Ground Truth for the Training Set Was Established
As there is no "training set" for an AI/ML algorithm in this context, this question is not applicable. The device relies on direct antigen detection via immunofluorescence, not on learned patterns from a training dataset.
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SECTION 05, 510(K) SUMMARY
Applicant:
DIAGNOSTIC HYBRIDS, INC. 1055 East State Street Suite 100 Athens, OHIO 45701
Contact Information:
Gail R. Goodrum Vice President, Regulatory Affairs E-mail: goodrum(@dhiusa.com Telephone: 740-589-3300 FAX: 740-593-8437
Date of preparation of 510(k) summary:
December 22, 2008
Device Name:
Trade name - D3 DFA Metapneumovirus Identification Kit Common name - Fluorescent antibody test for detecting human metapneumovirus Classification name - (blank) Product Code - OMG Regulation - 21 CFR 866.3980, Class II, Respiratory viral panel multiplex nucleic acid assay reagents; Panel Microbiology (83)
Legally marketed device to which equivalence is claimed:
K082688, Pro hMPV+ Assay
Intended Use: The Pro hMPV+ Assay is a Real Time RT-PCR in vitro diagnostic test for the qualitative detection of human Metapneumovirus (hMPV) nucleic acid isolated and purified from nasopharyngeal swab (NP) specimens obtained from individuals exhibiting signs and symptoms of acute respiratory infection. This assay targets a highly conserved region of the Nucleocapsid gene of hMPV. The detection of hMPV nucleic acid from symptomatic patients aids in the diagnosis of human respiratory hMPV infection if used in conjunction with other clinical and laboratory findings. This test is not intended to differentiate the four genetic sub-lineages of hMPV.
Negative results do not preclude hMPV infection and should not be used as the sole basis for diagnosis, treatment or other management decisions.
6 2009 MAR
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Diagnostic Hybrids, Inc.
D3 DFA METAPNEUMOVIRUS IDENTIFICATION KIT
Device Description:
The D DFA Metapneumovirus Identification Kit uses a blend of three hMPV antigen-specific murine MAbs that are directly labeled with fluorescein for detection of hMPV. The reagent detects but does not differentiate between the four recognized subtypes of hMPV (subtypes A1, A2, B1, and B2).
Kit Components:
-
- Metapneumovirus DFA Reagent, 5-mL. One dropper bottle containing a blend (see below for MAb discussion) of fluorescein-labeled murine monoclonal antibodies directed against MPV. The buffered, stabilized, aqueous solution contains Evans Blue as a counter-stain and 0.1% sodium azide as a preservative.
-
- hMPV Antigen Control Slides, 5 slides. Five individually packaged control slides, each with a well containing cell culture-derived MPV positive cells and a well containing cell culture-derived negative cells. Each slide is intended to be stained only one time. Control material has been treated to be non-infectious; however normal laboratory precautions are required when the material is handled.
-
- 40X PBS Concentrate, 25-mL. One bottle containing a 40X concentrate consisting of 4% sodium azide (0.1% sodium azide after dilution to 1X using de-mineralized water) in PBS.
-
- Mounting Fluid, 7-mL. One dropper bottle containing an aqueous, buffer-stabilized solution of glycerol and 0.1% sodium azide.
The cells to be tested, derived from a clinical specimen or cell culture, are placed onto a glass slide, allowed to air dry and are fixed in acetone. The Metapneumovirus DFA Reagent is added to the cells which are then incubated for 15 to 30 minutes at 35℃ to 37℃ in a humidified chamber or humidified incubator. The stained cclls are then washed with the diluted phosphate buffered saline (PBS), a drop of the supplied Mounting Fluid is added and a coverslip is placed on the prepared cells. The cells are examined using a fluorescence microscope. The hMPV infected cells will fluoresce apple-green. Uninfected cells will contain no fluorescence but will be stained red by the Evans Blue counter-stain.
It is recommended that specimens found to contain no fluorescent cells after examination of the direct specimen be confirmed by an FDA cleared hMPV molecular assay.
Intended Use:
The Diagnostic Hybrids, Inc. device, D3 DFA Metapneumovirus Identification Kit, is intended for the qualitative detection and identification of human metapneumovirus (hMPV) in nasal and nasopharyngeal swabs and aspirates/washes or cell culture. The assay detects hMPV antigens by immunofluorescence using a
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Diagnostic Hybrids, Inc.
D3 DFA METAPNEUMOVIRUS IDENTIFICATION KIT
02/27/2009 Section 5 - Page 3 of 13
blend of three monoclonal antibodies (MAbs), from patients with signs and symptoms of acute respiratory infection. This assay detects but is not intended to differentiate the four recognized genetic sub-lineages of hMPV.
Negative results do not preclude hMPV infection and should not be used as the sole basis for diagnosis, treatment or other management decisions. It is recommended that specimens found to be negative after examination of the direct specimen results be confirmed by an FDA cleared hMPV molecular assay.
Technological Characteristics, Compared to Predicate Device:
| TABLE 1: | Technological Characteristics Comparison of Devices | ||
|---|---|---|---|
| D3 DFA Metapneumovirus IdentificationKit (Subject) | Pro hMPV+ Assay (Predicate) | DHI Human MetapneumovirusReal-Time, Reverse TranscriptionPCR Assay (Reference) | |
| Target: | |||
| Searches of the National Center forBiotechnology Information (NCBI)databasesa yielded presumptive evidence thatthe target for each of the 3 MAb clones is theMPV nucleoprotein. Nine proteins areknown to be encoded in the hMPV genome.bOf the nine proteins, only the nucleoproteinis of a size equivalent to the 46 kDa sizenoted on the Western of the 3 MAb clones. | The Pro hMPV+ Supermix containsoligonucleotide primers and target-specific oligonucleotide probes. Theprimers are complementary to highlyconserved regions of geneticsequences of the nucleocapsid ofhMPV. | The DHI Human MetapneumovirusReal-Time, Reverse TranscriptionPCR Assay master mix containsoligonucleotide primers and target-specific oligonucleotide probes. Theprimers are complementary to highlyconserved sequences within thenucleocapsid gene of hMPV. | |
| Specimen: | |||
| Nasal and nasopharyngeal swabs andaspirates or cell culture. | Nasopharyngeal swab specimensusing a polyester, rayon or nylontipped swab and placed into viraltransport medium | Nasal and nasopharyngeal swabs andaspirates or cell culture. | |
| Detection Methods: | |||
| The assay detects specific hMPV viralantigens by immunofluorescence usingmonoclonal antibodies (MAbs). The cells tobe tested, derived from a clinical specimen orcell culture, are placed onto a glass slide andallowed to air dry. The cells are fixed inacetone. The hMPV DFA reagent is added tothe cells which are then incubated for 15 to30 minutes at 35° to 37°C in a humidifiedchamber or humidified incubator. Thestained cells are then washed with the dilutedphosphate buffered saline (PBS), a drop ofthe supplied Mounting Fluid is added and acoverslip is placed on the prepared cells. Thecells are examined using a fluorescencemicroscope. The hMPV infected cells will | Reverse transcription of the RNA inthe sample into complementary DNA(cDNA) and subsequentamplification of DNA is performedin a Cepheid SmartCycler®IIinstrument. In this process, the probeanneals specifically to the templatefollowed by primer extension andamplification. The Pro hMPV+Assay is based on TaqManchemistry, which utilizes the 5' - 3'exonuclease activity of the Taqpolymerase to cleave the probe thusseparating the reporter dye from thequencher. This generates an increasein fluorescent signal upon excitation | Reverse transcription of the RNA inthe sample into complementary DNA(cDNA) and subsequent amplificationof DNA is performed in a StratageneMx3000p instrument. In this process,the probe anneals specifically to thetemplate followed by primer extensionand amplification. The DHI HumanMetapneumovirus Real-Time, ReverseTranscription Assay is based onTaqMan chemistry, which utilizes the5'-3' exonuclease activity of the Taqpolymerase to cleave the probe thusseparating the reporter dye from thequencher. This generates an increasein fluorescent signal upon excitation | |
| fluoresce apple-green. Uninfected cells will | from a light source. With each cycle, | from a light source. With each cycle. | |
| TABLE 1: | Technological Characteristics Comparison of Devices | ||
| D³ DFA Metapneumovirus IdentificationKit (Subject) | Pro hMPV+ Assay (Predicate) | DHI Human MetapneumovirusReal-Time, Reverse TranscriptionPCR Assay (Reference) | |
| contain no fluorescence but will be stainedred by the Evans Blue counter-stain. | additional reporter dye molecules arecleaved from their respective probes,further increasing fluorescent signal.The amount of fluorescence at anygiven cycle is dependent on theamount of amplification productspresent at that time. Fluorescentintensity is monitored during eachPCR cycle by the SmartCycler IIinstrument. | additional reporter dye molecules arecleaved from their respective probes,further increasing fluorescent signal.The amount of fluorescence at anygiven cycle is dependent on theamount of amplification productspresent at that time. Fluorescentintensity is monitored during eachPCR cycle by the Stratagene Mx3000pinstrument. | |
| Analytical sensitivity: | |||
| Analytical detection limits on directspecimens for the D³ DFAMetapneumovirus Identification Kitwere addressed using quantified culturesof characterized isolates of each of the 4recognized genetic sublineages of hMPV(A1, A2, B1, and B2). The infectedculture cells from a 1,000 infectedcells/mL culture were serially dilutedwith a suspension of uninfected LLC-MK2 cells. 25-µL aliquots from eachdilution level were spotted onto 10replicate microscope slides, then fixedand stained according to the instructionsfor use described in this product insert.Each cell spot was examined at 200xmagnification. Results were reported asnumbers of positive replicates for eachset of 10. Analytical detection limits foreach of the 4 hMPV genetic sublineageswere defined as the lowest dilutions atwhich at least 9 out of 10 replicates weredetected. Based on this testing the LoDfor each subtype was A1= 25 infectedcells/mL, A2 = 200 infected cells/mL, B1 =50 infected cells/ml., B2 = 100 infectedcells/mL.Detection limit on cell culture amplifiedspecimens of the D³ DFA MetapneumovirusIdentification Kit was addressed using a cellculture system. Analytical detection limits oncell culture amplified specimens for hMPVsubtypes A1, A2, B1, and B2 wereestablished with results reported in numbersof fluorescent cells per cell monolayer. Eachmaster stock virus preparation was diluted ina ten-fold manner. Eight wells of a 48-wellR-Mix cell culture plate were inoculated with | The analytical sensitivity (limit ofdetection or LoD) of the Pro hMPV+Assay was determined usingquantified (TCID₅₀/mL) cultures of 2hMPV (subtype A2 and subtype B2)strains serially diluted innasopharyngeal clinical matrix. Eachviral strain was extracted using theRoche MagNA Pure LC and tested inreplicates of 20 per concentration ofvirus. Analytical sensitivity (LoD) asdefined as the lowest concentration at which 3 95% of all replicates testedpositive, ranged from 102 to 101TCID₅₀/mL.D ConcentrationhMPV subtype A2 102 TCID₅₀/mLhMPV subtype B2 101 TCID₅₀/mL | Analytical validation of the real-time hMPV RT-PCR followed bybi-directional sequencing analysiscomparator assay includedanalytical sensitivity and reactivitystudy, analytical specificity study,and extraction efficiency study.The analytical sensitivity (limit ofdetection or LoD) of the real-timehMPV RT-PCR followed by bi-directional sequencing analysiscomparator assay was determinedusing quantified (TCID₅₀/mL)stocks of the 4 hMPV (subtypesA1, A2, B1 and B2) strains dilutedin hMPV negative nasopharyngealclinical matrix, and ranged from 10- 50 TCID₅₀/mL. | |
| TABLE 1: Technological Characteristics Comparison of Devices | |||
| D³ DFA Metapneumovirus Identification Kit (Subject) | Pro hMPV+ Assay (Predicate) | DHI Human Metapneumovirus Real-Time, Reverse Transcription PCR Assay (Reference) | |
| 0.2-mL volumes of each dilution. The plates were centrifuged at 700 xg for 60 minutes, and then incubated at 35°C to 37°C for 48-hours. Each well was stained with the D³ DFA Metapneumovirus Identification Kit then examined at 200x magnification and the number of fluorescent cells counted. In this study, the detection limit for the test on cell culture amplified specimens is defined as the lowest inoculum level at which positive wells (i.e. containing fluorescent cells) are observed, in terms of TCID₅₀. Table 1.1 below summarizes the results: | |||
| TABLE 1.1: Limit of Detection of the D³ DFA Metapneumovirus Identification Kit for Cell Culture Amplified Specimens (values are numbers of fluorescent staining cells per cell monolayer) | |||
| Virus Strain | Conc. of Inoculum | Fluorescent staining cells/well | |
| hMPV A1 | 50-TCID₅₀ | 47,39,41,31,26,30, 21,29 | |
| 5-TCID₅₀ | 0,0,0,3,1,0,2,0 | ||
| 0.5-TCID₅₀ | 0,0,0,0,0,0,0,0 | ||
| hMPV A2 | 50-TCID₅₀ | 10,13,23,13,23,15, 17,12 | |
| 5-TCID₅₀ | 3,1,1,4,2,2,0,0 | ||
| 0.5-TCID₅₀ | 0,0,0,0,0,0,0,0 | ||
| hMPV B1 | 50-TCID₅₀ | 36,56,23,41,28,29, 34,28 | |
| 5-TCID₅₀ | 4,7,0,3,1,0,4,4 | ||
| 0.5-TCID₅₀ | 0,0,0,0,0,0,0,0 | ||
| hMPV B2 | 50-TCID₅₀ | 25,49,36,41,53,68, 43,27 | |
| 5-TCID₅₀ | 0,3,1,1,5,6,3,5 | ||
| 0.5-TCID₅₀ | 0,0,0,0,0,0,0,0 | ||
| Analytical specificity (cross reactivity studies; various strains of microorganisms and cell lines): | |||
| Viruses | 59 | 26 | 14 |
| Bacteria | 25 | 21 | 0 |
| Chlamydia spp. | 3 | 2 | 0 |
| Yeast | 1 | 1 | 0 |
| Protozoan | 1 | 0 | 0 |
| TABLE 1: Technological Characteristics Comparison of Devices | |||
| D³ DFA Metapneumovirus IdentificationKit (Subject) | Pro hMPV+ Assay (Predicate) | DHI Human MetapneumovirusReal-Time, Reverse TranscriptionPCR Assay (Reference) | |
| Cell lines | 17 | 0 | 0 |
| Human genomicDNA | 1 | ||
| Human total RNA | 1 |
ª NCBI (National Center for Biotechnology Information) internet web site http://www.ncbi.nlm.nih.gov/
DHI-MPV_Sec05_510k-Summary_09FEB27_Corrected.doc
8 Proteins encoded in hMPV genome are nucleoprotein, matrix protein, fusion glycoprotein precursor, matrix protein M2-1, matrix protein M2-2, small hydrophobic protein attachment glycoprotein G, RNA dependent RNA polymerase.
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DHI-MPV_Sec05_510k-Summary_09FEB27_Corrected.doc
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02/27/2009 Section 5 - Page 6 of 13
Analytical Performance:
The MAbs all recognize a hMPV protein, approximately 46 kiloDaltons in size, which corresponds with the size of hMPV nucleoprotein, but do not compete with one another for binding sites (as demonstrated by SDS-PAGE analyses, against lysates of cell culture infected with hMPV subtypes A1, A2, B1, and B2).
Analytical specificity of the MPV DFA Reagent was evaluated against other respiratory viruses (multiple strains of adenovirus, influenza A, influenza B, respiratory syncytial virus, and parainfluenza types 1, 2, and 3), as well as strains of 8 other viruses, and 30 other microorganisms that could be encountered in a respiratory specimen, cell culture contamination, or laboratory processing.
Reproducibility
Assay reproducibility was assessed at 3 laboratory sites with a panel of proficiencylevel antigen control slides. The reproducibility panel consisted of 5 panel members. Each panel member was a 2-well slide spotted with the same cell preparation in each well. The cell preparations used to construct the slides are the following:
-
- Non-infected LLC-MK2 cells.
-
- Low level hMPV (A1 strain) grown in LLC-MK2 cells (manufactured to contain between 4 to 10% infected cells).
-
- Mid level hMPV (A1 strain) grown in LLC-MK2 cells (manufactured to contain between 20 to 30% infected cells).
-
- High level hMPV (A1 strain) grown in LLC-MK2 cells (manufactured to contain between 50 to 75% infected cells).
Each panel was tested daily in two separate runs for 5-days by three different laboratories (30 total runs). The panel members were randomized with different slide identification numbers to act as a "blinded" panel. An hMPV Antigen Control Slide (two-well slide, one well contains cell culture-derived hMPV positive cells and one well contains cell culture-derived negative cells) was stained according to the D-DFA Metapneumovirus Identification Kit instructions for use with each run. The following results were recorded for both the control slides and the panel slides:
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02/27/2009 Section 5 - Page 7 of 13
-
- Presence or absence of green fluorescence.
-
- Percent of cells exhibiting green fluorescence.
A single lot of D MPV Kit was used. A total of 210 data points were included in the reproducibility study data analysis (7 samples and controls/run X 2 runs/day X 5 days X 3 sites = 210). The combined data from the three sites demonstrated that the detection of hMPV occurs in a reproducible manner. The presence of hMPV infected cells was reported in 100% (120/120) of the wells in which infected cells were present. The combined data from the three sites also demonstrated that no hMPV was detected in non-infected cells. The absence of hMPV was reported in 100% (90/90) of the wells in which infected cells were not present. The total percent agreement for the D' DFA Metapneumovirus Identification Kit was 100% (210/210). The Table below summarizes the reproducibility study results:
| Reproducibility Study Results | ||||||||
|---|---|---|---|---|---|---|---|---|
| PanelMember | hMPV A1Low Level | hMPV A1Mid Level | hMPV A1High Level | hMPV A1Negative | PositiveControl | NegativeControl | Total %Agreement | |
| Concentration | 4 to 10%infectedcells | 20 to 30%infectedcells | 50 to 75%infectedcells | Non-infectedcells | 50 to 75%infectedcells | Non-infectedcells | ||
| Site 1 | Agreement withExpected result | 10/10(100%) | 10/10(100%) | 10/10(100%) | 20/20(100%) | 10/10(100%) | 10/10(100%) | 70/70(100%) |
| Site 2 | Agreement withExpected result | 10/10(100%) | 10/10(100%) | 10/10(100%) | 20/20(100%) | 10/10(100%) | 10/10(100%) | 70/70(100%) |
| Site 3 | Agreement withExpected result | 10/10(100%) | 10/10(100%) | 10/10(100%) | 20/20(100%) | 10/10(100%) | 10/10(100%) | 70/70(100%) |
| Total Agreementwith Expectedresult | 30/30(100%) | 30/30(100%) | 30/30(100%) | 60/60(100%) | 30/30(100%) | 30/30(100%) | 210/210(100%) | |
| 95% CI | 88.4%-100% | 88.4%-100% | 88.4%-100% | 94.0%-100% | 88.4%-100% | 88.4%-100% | 98.3%-100% |
Clinical Performance:
Performance characteristics of the DHI D3 DFA Metapneumovirus Identification Kit testing direct respiratory specimens were established during prospective studies at 3 geographically diverse U.S. clinical laboratories during the 2005 and 2006 respiratory virus seasons (December 2005 - April 2006 and December 2006 -March 2007). All specimens used in the studies meeting the inclusion and exclusion criteria represented excess, remnants of respiratory specimens that were prospectively collected from symptomatic individuals suspected of respiratory infection, and were submitted for routine care or analysis by each site, and that otherwise would have been discarded. Individual specimens were delinked from all patient identifiers and given a study sample code. All clinical sites were granted waivers of informed consent by their IRBs for this study.
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02/27/2009 Section 5 - Page 8 of 13
Performance of the D3 DFA Metapneumovirus Identification Kit was assessed and compared to a predetermined algorithm that used composite comparator methods at clinical study site 1 and 2. The composite comparator methods consisted of viral culture and a validated real-time RT-PCR comparator assay targeting the hMPV nucleocapsid gene followed by bi-directional sequencing analysis". "True" hMPV positive was defined as any sample that either tested positive by viral culture, or had bi-directional sequencing data meeting pre-defined quality acceptance criteria that matched hMPV sequences deposited in the National Center for Biotechnology Information (NCBI) GenBank database (www.ncbi.nlm.nih.gov), with acceptable Evalues . "True" hMPV negative was defined as any sample that tested negative by both viral culture and the hMPV real-time RT-PCR comparator assay..
Performance of the D3 DFA Metapneumovirus Identification Kit at clinical study site 3 was evaluated and compared only to the validated hMPV real-time RT-PCR followed by bi-directional sequencing analysis comparator assay described above. Any sample that had bi-directional sequencing data meeting pre-defined quality acceptance criteria that matched hMPV sequences deposited in the National Center for Biotechnology Information (NCBI) GenBank database (www.ncbi.nlm.nih.gov), with acceptable E-values, was considered as hMPV positive, and the real-time hMPV RT-PCR comparator assay negatives were considered as hMPV negatives at this site.
Study Site 1
Study Site 1 evaluated a total of 1564 fresh respiratory specimens submitted. December 2006 through March 2007, to the laboratory for respiratory virus testing. Slides were prepared from Phosphate Buffered Saline (PBS)-washed cells from the fresh specimens and fixed according to the prescribed protocol. The slides were stained in accordance with the procedure in the product insert.
Therefore an E-Value ranging from 2e-67 to 2e-77 has a very low probability of occurring purely by chance.
DHI-MPV_Scc05_510k-Summary_09FEB27_Corrected.doc
6 Analytical validation of the real-time hMPV RT-PCR followed by bi-directional sequencing assay included analytical sensitivity and reactivity study, analytical specificity study, and extraction efficiency study. The analytical sensitivity (limit of detection or LoD) of the real-time hMPV RT-PCR followed by bi-directional sequencing analysis comparator assay was determined using quantified (TCIDs) mL) stocks of the 4 hMPV (subtypes A1, A2, B1 and B2) strains diluted in hMPV negative nasopharyngeal clinical matrix, and ranged from 10 - 50 TCIDsofmL.
d The E-values generated from the clinical trials range from a low of 2e-67. The E-Value from NCBI BLAST Alignment indicates the statistical significance of a given pair-wise alignment and reflects the size of the database and the scoring system used. The lower the li-Value, the more significant the hit. A sequence alignment that has an E-Value of 1c-3 means that this similarity has a 1 in 1000 chance of occurring by chance alone. (http://www.ncbi.nlm.nih.gov/books/bv.fcgi'?rid=handbook.section.614}.
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02/27/2009 Section 5 - Page 9 of 13
| Sex | F | M |
|---|---|---|
| Total | 687 | 877 |
| Age: <1m | 42 | 50 |
| ≥ 1m to < 2y | 444 | 617 |
| ≥2y to <12y | 164 | 185 |
| ≥ 12y to < 18y | 30 | 20 |
| ≥ 18y to < 21y | 4 | 3 |
| ≥ 21y | 3 | 2 |
| Age Not Reported | 0 | 0 |
Table 2 shows the age and gender distribution for individuals studied at study site 1:
Of the 1564 fresh respiratory specimens tested, 1509 were nasal wash/nasopharyngeal aspirate specimens. Due to insufficient sample numbers to establish performance of the D' DFA Metapneumovirus Identification Kit, 55 other types of respiratory specimens were removed from performance analysis. Of the 1509 fresh nasal wash/nasopharyngeal aspirate specimens tested, 27 were further excluded from the performance analysis due to insufficient volume for the comparator methods, resulting in a total of 1482 fresh nasal wash/nasopharyngeal aspirate specimens for analysis. Table 3 below shows the study results of the claimed specimen type at study site 1:
| TABLE 3: Study Site 1- Comparison of Results using D3 DFA MPVKit, with Results using the Composite Comparator Methods | |||
|---|---|---|---|
| Fresh NasalWash/NasopharyngealAspirate | Composite Comparator Methods | ||
| DHI DSFA | Positive | Negative | Total |
| Positive | 122 | 3 | 125 |
| Negative | 108 | 1249 | 1357 |
| Total | 230 | 1252 | 1482 |
| 95% CI | |||
| Sensitivity | 122/230 | 53.0% | 46.6%-59.5% |
| Specificity | 1249/1252 | 99.8% | 99.3%-99.9% |
Study Site 2
Study Site 2 evaluated a total of 371 fresh respiratory specimens submitted, December 2005 through January 2006, to the laboratory for respiratory virus testing. Slides were prepared from Phosphate Buffered Saline (PBS)-washed cells from the fresh specimens and fixed according to the prescribed protocol. The slides were stained in accordance with the procedure in the product insert.
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02/27/2009 Section 5 - Page 10 of 13
Table 4 below shows the age and gender distribution for individuals studied at study site 2:
| TABLE 4: Site 2 – Age and Gender Distribution | ||
|---|---|---|
| Sex | F | M |
| Total | 155 | 216 |
| Age: <1m | 2 | 5 |
| ≥ 1m to < 2y | 50 | 83 |
| ≥2y to <12y | 26 | 37 |
| ≥ 12y to < 18y | 2 | 5 |
| ≥ 18y to < 21y | 1 | 0 |
| ≥ 21y | 74 | 86 |
| Age Not Reported | 0 | 0 |
Of the 371 fresh respiratory specimens tested, all were nasal/nasopharyngeal swab specimens. 3 were excluded from the performance analysis due to insufficient volume for the comparator methods, resulting in a total of 368 fresh nasal/nasopharyngeal swab specimens for analysis. Table 5 below shows the study results of the claimed specimen type at study site 2:
| TABLE 5: Study Site 2- Comparison of Results using D³ DFA MPVKit, with Results using the Composite Comparator Methods | |||
|---|---|---|---|
| Fresh Nasal/NasopharyngealSwab | Composite Comparator Methods | ||
| DHI DSFA | Positive | Negative | Total |
| Positive | 41 | 1 | 42 |
| Negative | 17 | 309 | 326 |
| Total | 58 | 310 | 368 |
| Sensitivity | 41/58 | 70.7% | 57.3%-81.9%95% CI |
| Specificity | 309/310 | 99.7% | 98.2%-100% |
Study Site 3
Study Site 3 evaluated a total of 174 fresh respiratory specimens submitted. March 2006 through April 2006, to the laboratory for respiratory virus testing. Slides were prepared from Phosphate Buffered Saline (PBS)-washed cells from the fresh specimens and fixed according to the prescribed protocol. The slides were stained in accordance with the procedure in the product insert.
Table 6 below shows the age and gender distribution for individuals studied at study site 3:
DHI-MPV_Sec05_510k-Summary_09FFB27_Corrected.doc
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02/27/2009 Section 5 - Page 11 of 13
| TABLE 6: Site 3 – Age and Gender Distribution | |||
|---|---|---|---|
| Sex | F | M | Sex Not Reported |
| Total | 78 | 95 | 1 |
| Age: <1m | 1 | 1 | 0 |
| ≥ 1m to < 2y | 19 | 37 | 0 |
| ≥2y to <12y | 16 | 17 | 0 |
| ≥ 12y to < 18y | 3 | 6 | 0 |
| ≥ 18y to < 21y | 2 | 0 | 0 |
| ≥ 21y | 26 | 22 | 0 |
| Age Not Reported | 11 | 12 | 1 |
Of the 174 fresh respiratory specimens tested, 62 were nasal wash/nasopharyngeal aspirate specimens, and 110 were nasal/nasopharyngeal swab specimens. Of the 62 nasal wash/nasopharyngeal aspirate specimens, 30 were excluded from the performance analysis due to insufficient volume for the comparator method, resulting in a total of 32 fresh nasal wash/nasopharyngeal aspirate specimens for analysis. Of the 110 nasal/nasopharyngeal swab specimens, 44 were excluded from the performance analysis due to insufficient volume for the comparator method, resulting in a total of 66 fresh nasal/nasopharyngeal swab specimens for analysis. Table 7 and 8 below show the study results of the claimed specimen types at study site 3:
| TABLE 7: Study Site 3- Comparison of Results using D³ DFA MPV Kit, withResults using the hMPV real-time RT-PCR followed by bi-directionalsequencing analysis comparator assay | |||
|---|---|---|---|
| Fresh NasalWash/NasopharyngealAspirate | Comparator Assay | ||
| DHI DSFA | Positive | Negative | Total |
| Positive | 9 | 0 | 9 |
| Negative | 0 | 23 | 23 |
| Total | 9 | 23 | 32 |
| 95% CI | |||
| Positive Percent Agreement* | 9/9 | 100.0% | 66.4%-100% |
| Negative Percent Agreement* | 23/23 | 100.0% | 85.2%-100% |
| TABLE 8: Study Site 3- Comparison of Results using D³ DFA MPV Kit, withResults using the hMPV real-time RT-PCR followed by bi-directionalsequencing analysis comparator assay | |||
|---|---|---|---|
| Fresh Nasal/NasopharyngealSwab | Comparator Assay | ||
| DHI DSFA | Positive | Negative | Total |
| Positive | 3 | 0 | 3 |
| Negative | 1 | 62 | 63 |
| Total | 4 | 62 | 66 |
| 95% CI |
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02/27/2009 Section 5 - Page 12 of 13
| Positive Percent Agreement* | 3/4 | 75.0% | 19.4%-99.4% |
|---|---|---|---|
| Negative Percent Agreement* | 62/62 | 100.0% | 94.2%-100% |
- Since the performance of the D3 DFA Metapneumovirus Identification Kit at clinical study site 3 was not assessed against the predetermined comparator methods, positive and negative percent agreements, instead of sensitivity and specificity, are used in the performance presentation.
Cultured Cells Testing
Performance characteristics of the DHI D DFA Metapneumovirus Identification Kit testing cultured cell specimens were established during a prospective study at DHI during the 2007 respiratory virus seasons (January - April 2008). All specimens used in the studies meeting the inclusion and exclusion criteria represented excess. remnants of respiratory specimens that were prospectively collected from symptomatic individuals suspected of respiratory infection, and were submitted for routine care or analysis by each collection site, and that otherwise would have been discarded. Individual specimens were delinked from all patient identifiers and given a study sample code.
Performance of the D3 DFA Metapneumovirus Identification Kit testing cultured cell specimens was evaluated and compared to the same validated hMPV real-time RT-PCR followed by bi-directional sequencing analysis comparator assay as described earlier, at clinical study site 4. Any cultured cell specimen that had bidirectional sequencing data meeting pre-defined quality acceptance criteria that matched hMPV sequences deposited in the National Center for Biotechnology Information (NCBI) GenBank database (www.ncbi.nlm.nih.gov), with acceptable Evalues, was considered as hMPV positive, and the real-time hMPV RT-PCR comparator assay negative cultured cell specimens were considered as hMPV negatives.
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02/27/2009 Section 5 - Page 13 of 13
Study Site 4
·
- Study Site 4 evaluated a total of 74 freeze-thawed nasopharyngeal swab specimens that were cultured and stained in accordance with the D3 DFA Metapneumovirus Identification Kit procedure. Table 9 below shows the study results testing cultured cell specimens at study site 4:
| TABLE 9: Study Site 4- Comparison of Results using D³ DFA MPV Kit, withResults using the hMPV real-time RT-PCR followed by bi-directionalsequencing analysis comparator assay | |||
|---|---|---|---|
| Freeze-thawedNasopharyngeal SwabAmplified in Cell Culture | DHI hMPV RT-PCR Followed by SequencingComparator Assay | ||
| DHI DFA | Positive | Negative | Total |
| Positive | 5 | 0 | 5 |
| Negative | 1 | 68 | 69 |
| Total | 6 | 68 | 74 |
| 95% CI | |||
| Positive Percent Agreement | 5/6 | 83.3% | 35.9%-99.6% |
| Negative Percent Agreement | 68/68 | 100.0% | 99.7%-100% |
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DEPARTMENT OF HEALTH & HUMAN SERVICES
Image /page/13/Picture/1 description: The image contains the text 'Public Health Service'. The text is in a simple, sans-serif font and is horizontally aligned. The words are stacked on top of each other.
Food and Drug Administration 2098 Gaither Road Rockville MD 20850
Ms. Gail Goodrum Vice President, Regulatory Affairs Diagnostic Hybrids 1055 East State Street Suite 100 Athens, OH 45701
Re: K090073
Trade/Device Name: D3 DFA Metapneumovirus Identification Kit Regulation Number: 21 CFR 866.3980 Regulation Name: Respiratory viral panel multiplex nucleic acid assay Regulatory Class: Class II Product Code: OMG Dated: December 22, 2008 Received: January 12, 2009
6 2009
MAR
Dear Ms. Goodrum:
We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration.
If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to such additional controls. Existing major regulations affecting your device can be found in Title 21, Code of Federal Regulations (CFR), Parts 800 to 895. In addition, FDA may publish further announcements concerning your device in the Federal Register.
Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Parts 801 and 809); and good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820).
__, and I am not going to apologize for it.
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Page 2 -
This letter will allow you to begin marketing your device as described in your Section 510(k). premarket notification. The FDA finding of substantial equivalence of your device to a legally marketed predicate device results in a classification for your device and thus, permits your device to proceed to the market.
If you desire specific advice for your device on our labeling regulation (21 CFR Part 801), please contact the Office of In Vitro Diagnostic Device Evaluation and Safety at 240-276-0450. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21CFR Part 807.97). For questions regarding postmarket surveillance, please contact CDRH's Office of Surveillance and Biometric's (OSB's) Division of Postmarket Surveillance at 240-276-3474. For questions regarding the reporting of device adverse events (Medical Device Reporting (MDR)), please contact the Division of Surveillance Systems at 240-276-3464. You may obtain other general information on your responsibilities under the Act from the Division of Small Manufacturers, International and Consumer Assistance at its toll-free number (800) 638-2041 or (240) 276-3150 or at its Internet address http://www.fda.gov/cdrh/industry/support/index.html.
Sincerely vours.
Sally attaym
Sally A. Hojvat, M.Sc., Ph.D. Director Division of Microbiology Devices Office of In Vitro Diagnostic Device Evaluation and Safety Center for Devices and Radiological Health
Enclosure
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Indication for Use
510(k) Number (if known): K090073
Device Name: D DFA Metapneumovirus Identification Kit
Indication For Use:
The Diagnostic Hybrids, Inc. device, Dr DFA Metapneumovirus Identification Kit, is intended for the qualitative detection and identification of human metapneumovirus (hMPV) in nasal and nasopharyngeal swabs and aspirates/washes or cell culture. The assay detects hMPV antigens by immunofluorescence using a blend of three monoclonal antibodies (MAbs), from patients with signs and symptoms of acute respiratory infection. This assay detects but is not intended to differentiate the four recognized genetic sublineages of hMPV.
Negative results do not preclude hMPV infection and should not be used as the sole basis for diagnosis, treatment or other management decisions. It is recommended that specimens found to be negative after examination of the direct specimen results be confirmed by an FDA cleared hMPV molecular assay.
Prescription Use X And/Or (21 CFR Part 801 Subpart D)
Over the Counter Use (21 CFR Part 801 Subpart C)
(PLEASE DO NOT WRITE BELOW THIS LINE; CONTINUE ON ANOTHER PAGE IF NEEDED)
Concurrence of CDRH, Office of In Vitro Diagnostic Device Evaluation and Safety (OVD)
Uwe Schof
Division Sign-Off Office of In Vitro Diagnostic Device Evaluation and Safety
510(k) K090073
§ 866.3980 Respiratory viral panel multiplex nucleic acid assay.
(a)
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.
(b)
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.