DIAGNOSTIC HYBRIDS D3 DFA VARICELLA-ZOSTER VIRUS IDENTIFICATION KIT, 01-020000

{0} 1 # 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY ONLY TEMPLATE A. 510(k) Number: K070206 B. Purpose for Submission: The 510(k) holder would like to introduce D³ DFA Varicella Zoster Virus Identification Kit into interstate commerce. C. Measurand: Varicella zoster antigen D. Type of Test: Determination of the presence of varicella zoster in amplified cell culture. E. Applicant: Diagnostic Hybrids, Inc. F. Proprietary and Established Names: D³ DFA Varicella Zoster Virus Identification Kit. G. Regulatory Information: 1. Regulation section: 866.3900 2. Classification: II 3. Product code: GQW 4. Panel: 81 H. Intended Use: 1. Intended use(s): The Diagnostic Hybrids, Inc D³ DFA Varicella-zoster Virus Identification Kit is intended for use in the qualitative detection of varicella-zoster virus (VZV) in cell cultures by immunofluorescence using fluoresceinated monoclonal antibodies (MAbs). Negative results do not preclude an infection and should not be used as the sole basis for diagnosis, treatment or other management decision. Performance testing has not been done on direct patient specimen testing 2. Indication(s) for use: Same as Intended Use. 3. Special conditions for use statement(s): For prescription use only 4. Special instrument requirements: Fluorescence microscope with the correct filter combination for FITC (excitation peak = 490 nm, emission peak = 520nm). {1} I. Device Description: The Diagnostic Hybrids, Inc. D³ DFA Varicella-Zoster Identification Kit includes a blend of two fluorescein-labeled murine monoclonal antibodies directed against VZV antigens. The kit includes the following components which are used for screening and final virus identification in cell cultures inoculated with patient specimens. Kit Components: - VZV DFA Reagent. A blend of two fluorescein labeled murine monoclonal antibodies directed against a recombinant glycoprotein E (gE) from the Ellen strain of VZV. The buffered, stabilized, aqueous solution contains Evan’s Blue as a counterstain and 0.1% sodium azide as preservative. - VZV Antigen Control Slides. Individually packaged control slides containing wells with cell culture derived positive and negative control cells. Each VZV Positive well is identified. The Negative wells contain uninfected cells. Each slide is intended for single use. - Wash Solution Concentrate. One bottle containing a 40X concentrate consisting of 4% sodium azide (after dilution to 1X with water, the concentration of sodium azide in the solution is 0.1%) in Phosphate Buffered Saline (PBS). - Mounting Fluid. An aqueous, buffered, stabilized solution of glycerol and 0.1% sodium azide. J. Substantial Equivalence Information: 1. Predicate device name(s): 1. Light Diagnostics Varicella-zoster (VZV) Direct Immunofluorescence Assay (DFA). 2. Predicate K number(s): K951799 3. Comparison with predicate: a Similarities: The similarities to predicate devices are in indicated use, operating principle, materials and formulation. | Similarities | | | | --- | --- | --- | | Item | Device | Predicate | | Intended Use | The Diagnostic Hybrids, Inc. D³ DFA Varicella-Zoster Virus Identification Kit is intended for use in the qualitative detection of recombinant glycoprotein E (gE) from the Ellen strain of VZV in cultures by using fluoresceinated monoclonal antibodies (MAb’s). | 1. The Light Diagnostics Varicella-zoster (VZV) Direct Immunofluorescence Assay (DFA) is intended for the qualitative detection of GPI and immediate early antigen of VZV from vesicular lesions. The kit is intended for use in culture confirmation with standard tube cultures and shell vials. | | Basic principle | DFA (Direct Fluorescent Antibody) test - | DFA (Direct Fluorescent Antibody) test - | {2} | Similarities | | | | --- | --- | --- | | Item | Device | Predicate | | | Immunofluorescence using fluoresceinated monoclonal antibodies in culture to include standard tube, shell vials and multiwell plates. | Immunofluorescence using fluoresceinated monoclonal antibodies in culture to include standard tube and shell vials. | | Antibody | Blend of murine monoclonal antibodies (MAbs) directed against two antigenic sites on the VZV recombinant protein, glycoprotein E. | Blend of murine monoclonal antibodies (MAbs) directed against two antigens, glycoprotein I and the immediate early antigen of VZV. | | Instrumentation (required but not provided) | Fluorescence microscope with the correct filter combination for FITC (excitation peak = 490 nm, emission peak = 520nm). | Fluorescence microscope with the correct filter combination for FITC (excitation peak = 490 nm, emission peak = 520nm). | | Sample type | Swabs of lesion specimens | Swabs of lesion specimens | b Differences: | Differences | | | | --- | --- | --- | | Item | Device | Predicate | | Procedural | Immunofluorescence testing following amplification in cell culture only. | Results considered presumptive for identification of VZV from direct patient specimens using fluoresceinated monoclonal antibodies. | K. Standard/Guidance Document Referenced (if applicable): N/A L. Test Principle: The test kit uses viral antigen-specific murine monoclonal antibodies that are directly labeled with fluorescein for rapid detection and identification of VZV which are directed against two antigenic sites on the VZV recombinant protein, glycoprotein E. The cell cultures are fixed in acetone. The VZV DFA Reagent is added to the monolayers in cell culture to determine the presence of viral antigens. After incubating, the infected cells are stained with a fluorescein conjugated MAb while uninfected cells and are counterstained with an Evan’s Blue dye. The stained cells are subsequently rinsed, overlaid with a drop of supplied mounting fluid, and the monolayer is protected with a coverslip. Cells are then examined using a fluorescence microscope. Interpretation of results: A result is considered positive when a cell cytoplasm and/or nucleus presents with a finely granular bright apple-green fluorescence. Uninfected cells will stain dull red due to the Evan’s Blue counter-stain which is included with this device. M. Performance Characteristics: {3} # 1. Analytical performance: a. Precision/Reproducibility: Not applicable b. Linearity/assay reportable range: Not applicable c. Traceability, Stability, Expected values (controls, calibrators, or methods): Not applicable d. Detection limit: VZV was diluted to a value of $357\text{-TCID}_{50}$ and serial 2-fold dilutions were then made to a final value of $1\text{-TCID}_{50}$ . Each dilution of virus was inoculated into 6 shell vials of H&amp;V Mix or MRC-5 cells, centrifuged at $700\mathrm{xg}$ for 60 minutes and incubated at $35 - 37^{\circ}\mathrm{C}$ for 48 hours. The Subject Kit or the Predicate Kit was used to stain 3 shell vials of each viral dilution according to the product inserts. The sensitivity of both fluorescent antibody stains is equivalent, with $\sim 1.4-$ to $5.7\text{-TCID}_{50}$ as the minimum viral dilution detected. e. Analytical specificity: See results for Cross-reactivity below. f. Assay cut-off: Not applicable # 2. Comparison studies: a Method comparison with predicate device: This study included two hundred and fifty-four (254) prospectively collected specimens submitted for VZV culture. Each specimen was evaluated by the $\mathrm{D}^3$ DFA VZV Identification Kit and compared to a currently marketed VZV identification kit. A combination of fresh (61) and frozen (193) specimens were tested. The numbers of fresh and frozen specimens tested are summarized below. | Number of Fresh vs. Frozen Specimens | | | | | --- | --- | --- | --- | | Site | Culture | | Site Total | | | Fresh | Frozen | | | 1 | 57 | 42 | 99 | | 2 | 1 | 31 | 32 | | 3 | 0 | 120 | 120 | # b Matrix comparison: Not applicable 3. Clinical studies: A total of (254) prospectively collected specimens were submitted for VZV culture. Three of the fresh specimens from Site 2 were toxic to cell culture and were not evaluated by either test. One specimen from Site 3 was negative in the multi-well plate culture, but was positive in the tube culture 10-days post inoculation. These evaluations were conducted at two external laboratory sites and one in-house laboratory: (1) A reference laboratory in the Southeastern United States; (2) A reference laboratory in the Southwestern United States; and (3) Diagnostic Hybrids, Inc in-house virology laboratory. Percent Agreement between the $\mathrm{D}^3$ DFA VZV and comparator tests was calculated {4} and tabulated for all the tested specimens is presented below. ## Percent Agreement of All Tests | D³ DFA VZV | Comparison Device | | | | --- | --- | --- | --- | | | + | - | | | | + | 42 | 1 | | | - | 0 | 208 | | Positive Percent Agreement (PPA) | 100% | | | | 95% CI- PPA | 91.6% to 100% | | | | Negative Percent Agreement (NPA) | 99.5% | | | | 95% CI - NPA | 97.3% to 99.9% | | | ## a Clinical Sensitivity: i Study Site 1: A total of 99 specimens were cultured for VZV using multi-well plates. Briefly, two hundred microliters (200-μL) of each specimen were inoculated using one well per specimen. The inoculated cells were centrifuged at 700xg for 60-minutes, incubated at 35° to 37°C for up to 72-hours then stained in accordance with each respective product insert (DHI and comparison device). DHI D³ DFA VZV Test and Comparison Device in multi-well plates. | D³ DFA VZV | Comparison Device | | | | --- | --- | --- | --- | | | + | - | | | | + | 22 | 0 | | | - | 0 | 77 | | PPA = | Agreed | 95% CI | | | | 100% | 84.6% to 100% | | | NPA = | 100% | 95.2% to 100% | | ii Study Site 2: A total of 35 specimens were cultured for VZV. Three fresh specimens from this site were toxic to cell culture and were not evaluated. Briefly, 200-μL from the specimens was inoculated into duplicate shell vials. The inoculated cells were incubated at 35° to 37°C for 72-hours then stained in accordance with the respective product insert (DHI and comparison devices). All calculations for confidence intervals were done according to the Exact Method Error! Bookmark not defined. The results of this testing are summarized below: {5} DHI D³ DFA VZV Test and Comparison Device in shell vials. | | Comparison Device | | | | --- | --- | --- | --- | | | | + | - | | D³ DFA VZV | + | 11 | 1 | | | - | 0 | 20 | | PPA = NPA = | Agreed | 95% CI | | | | 100% | 71.5% to 100% | | | | 95.1% | 76.2% to 99.9% | | iii Study Site 3: A total of 120 specimens were cultured for VZV. Briefly, two hundred microliters (200-μL) of each specimen was inoculated into one well per specimen of a multi-well plate and a single cell culture tube. The inoculated plates were centrifuged at 700xg for 60-minutes, incubated at 35° to 37°C for 72-hours then stained in accordance with each respective product insert (DHI and comparison devices). The results of this testing are summarized in Table 8a. The inoculated tubes were read for CPE daily for 14-days. Tubes exhibiting CPE were scraped and cell spots made on multiwell slides according to the comparison device's product insert procedure (the same procedure was used for both the DHI and the comparison devices). Tubes exhibiting no CPE at 14-days were also scraped and cell spots made to confirm the absence of VZV. The cell spots were fixed with acetone in accordance with each respective product insert (DHI and comparison device). The results of testing at this site, in house, are summarized in the two tables below. DHI D³ DFA VZV Test and Comparison Device in multi-well plates | | Comparison Device | | | | --- | --- | --- | --- | | | | + | - | | D³ DFA VZV | + | 8 | 0 | | | - | 0 | 112 | | PPA = NPA = | Agreed | 95% CI | | | | 100% | 63.1% to 100% | | | | 100% | 96.8% to 100% | | {6} 7 # DHI D³ DFA VZV Test and Comparison Device in tube cultures. | | Comparison Device | | | | --- | --- | --- | --- | | | | + | - | | D³ DFA VZV | + | 9 | 0 | | | - | 0 | 111 | | PPA = | Agreed | 95% CI | | | | 100% | 66.4% to 100% | | | NPA = | 100% | 96.7% to 100% | | ## a. Clinical specificity: **Cross reactivity testing:** This device was tested for cross-reactivity against a wide variety of cells and microorganisms compared to the predicate. No cross-reactivity was observed for 55 virus strains (cultured and processed for staining) or for 20 host culture cell types. Twenty-seven (27) bacterial cultures and one (1) yeast culture were stained and examined for cross-reactivity, including *Staphylococcus aureus*, a protein-A-producing bacterium. Staining of *S. aureus* appeared as small points of fluorescence while all other bacterial cultures were negative. [See Tables below for cross-reactivity study results.] Stringent conditions for cross-reactivity testing were achieved by using a high concentration of this device and relatively high titers of microorganisms. The DFA Reagent was prepared at 1.5X the concentration that is provided in the kit. - Fifty-five (55) virus strains were tested for cross reactivity. Depending on the particular virus, 150 to 2100 TCID₅₀ viruses were inoculated into a shell vial culture and incubated for 24 to 48 hours, to yield a 1+ to 3+ infection, processed and stained with the 1.5X DFAs according to the procedure detailed in the product insert. No cross reactivity was observed for the viruses listed below. {7} Virus Strains Tested for Cross Reactivity with VZV DFA Reagent | Organism | Strain or Type | Inoculum Concentration (TCID_{50}) | | --- | --- | --- | | Adenovirus | Type 1 | 350 | | Adenovirus | Type 5 | 350 | | Adenovirus | Type 6 | 350 | | Adenovirus | Type 7 | 350 | | Adenovirus | Type 8 | 350 | | Adenovirus | Type 10 | 350 | | Adenovirus | Type 14 | 350 | | Adenovirus | Type 18 | 350 | | Adenovirus | Type 31 | 350 | | Influenza A | Aichi | 2,100 | | Influenza A | Mal | 2,100 | | Influenza A | Hong Kong | 2,100 | | Influenza A | Denver | 2,100 | | Influenza A | Port Chalmers | 2,100 | | Influenza A | Victoria | 2,100 | | Influenza A | PR | 2,100 | | Influenza B | Hong Kong | 350 | | Influenza B | Maryland | 350 | | Influenza B | Mass | 350 | | Influenza B | Taiwan | 350 | | Influenza B | GL | 350 | | Influenza B | Russia | 350 | | Poliovirus | Type 1 | Commercially available slides stained^{Error!} Bookmark not defined. | | Poliovirus | Type 2 | | | Poliovirus | Type 3 | | | Epstein-Barr | Commercially available slides stained^{Error!} Bookmark not defined. | | | Rubeola | | | | Mumps | | | d Test material is from commercially available prepared slides. Each positive well contains 10 to 50% reactive cells. | Organism | Strain or Type | Inoculum Concentration (TCID_{50}) | | --- | --- | --- | | RSV | Long | 350 | | RSV | Wash | 350 | | RSV | 9320 | 350 | | Parainfluenza 1 | C-35 | Commercially available slides stained^{d} | | Parainfluenza 2 | Greer | | | Parainfluenza 3 | C 243 | | | HSV-1 | 1F | 150 | | HSV-1 | CWOH 0026 | 150 | | HSV-1 | CWOH 0015 | 150 | | HSV-1 | MacIntyre | 150 | | HSV-2 | MS | 150 | | HSV-2 | Strain G | 150 | | CMV | Towne | 700 | | CMV | Davis | 700 | | CMV | AD169 | 700 | | Echovirus | 4 | Commercially available slides stained^{d} Bookmark not defined. | | Echovirus | 6 | | | Echovirus | 9 | | | Echovirus | 11 | | | Echovirus | 30 | | | Echovirus | 34 | | | Coxsackievirus | B1 | Commercially available slides stained^{d} Bookmark not defined. | | Coxsackievirus | B2 | | | Coxsackievirus | B3 | | | Coxsackievirus | B4 | | | Coxsackievirus | B5 | | | Coxsackievirus | B6 | | {8} - Twenty (20) host culture cell types were tested for cross reactivity. Cell cultures were prepared in shell vial format. Confluent monolayers were stained with the 1.5X DFA Reagent according to the procedure as detailed in this product insert and then examined for cross reactivity. No cross reactivity was observed for the following cell lines presented below. ## Cell Lines Tested for Cross Reactivity using DHI the VZV DFA Reagent | A549 | NCI-H292 | | --- | --- | | BGMK | pCMK | | HEp-2 | pRhMK | | LLC-MK2 | pRK | | MDCK | RD | | MRC-5 | RhMK II | | MRHF | R-Mix | | Mv1Lu | Vero | | HFF | WI-38 | | McCoy | Vero 76 | - Twenty-eight microorganisms, including one (1) yeast culture and twenty-seven (27) bacterial cultures, were stained with the 1.5X DFA Reagent according to the procedure as detailed in this product insert, then examined for cross-reactivity. Except for Staphylococcus aureus, which was cross reactive with the VZV DFA Reagent (see above), all microorganisms tested negative. Concentrations for each bacterial organism cultured by DHI for cross reactivity testing were determined from suspensions of the bacteria in PBS by spectrophotometer according to McFarland standards of 1.0 and 2.0 (equaling approximately $3.0 \times 10^{6}$ and $6.0 \times 10^{6} \times 10^{4}$ CFU per mL). Slides were prepared with spots of $0.01\mathrm{-mL}$ of the suspensions to give either $3.0 \times 10^{4}$ or $6.0 \times 10^{4}$ per spot. At the same time, $1\mathrm{-mL}$ of each suspension was plated on an appropriate agar dish for colony confirmation. According to the confirmation agar cultures, initial concentrations of the bacterial organisms in the study ranged from $6.4 \times 10^{4}$ to $2.9 \times 10^{7}$ CFU. Results of testing are listed below. 9 {9} Bacteria and Yeast Tested for Cross Reactivity with VZV DFA Reagent | BACTERIA | CFU TESTED | | --- | --- | | Acinetobacter calcoaceticus | 9.7 x 10^{5} | | Bordetella bronchiseptica | 1.7 x 10^{5} | | Bordetella pertussis | 4.6 x 10^{6} | | Corynebacterium diphtheriae | 2.5 x 10^{6} | | Escherichia coli | 2.6 x 10^{5} | | Gardnerella vaginalis | 5.0 x 10^{5} | | Haemophilis influenzae type A | 9.3 x 10^{5} | | Klebsiella pneumoniae | 6.4 x 10^{6} | | Legionella pneumophila | 6.5 x 10^{4} | | Moraxella cartarrhalis | 6.4 x 10^{4} | | Neisseria gonorrhoeae | 1.3 x 10^{6} | | Proteus mirabilis | 2.1 x 10^{6} | | Pseudomonas aeruginosa | 1.0 x 10^{7} | | Salmonella enteriditis | 2.5 x 10^{6} | | Salmonella typhimurium | 1.7 x 10^{6} | | Staphylococcus aureus | 1.0 x 10^{7} | | Streptococcus agalactiae | 9.6 x 10^{6} | | Streptococcus pneumoniae | 8.0 x 10^{5} | | Streptococcus pyogenes | 2.9 x 10^{7} | | Acholeplasma laidlawi | ~6 x 10^{7} | | Mycoplasma hominis | ~6 x 10^{4} | | Mycoplasma orale | ~6 x 10^{4} | | Mycoplasma pneumoniae | ~6 x 10^{4} | | Mycoplasma salivarium | ~6 x 10^{7} | | Ureaplasma uralyticum | ~6 x 10^{4} | | Those listed below were procured as prepared slides: | Proportion of cells reactive | | Chlamydophila pneumoniae | 10 to 50% | | Chlamydia trachomatis | 10 to 50% | | YEAST | | | Candida glabrata | 8.7 x 10^{6} | c. Other clinical supportive data (when a. and b. are not applicable): Not applicable. 4. Clinical cut-off: Not applicable. 5. Expected values: The clinical studies used only specimens collected and cultured for the presence of VZV. Most of the specimen types used in the clinical studies was swabs taken from skin lesions (with two taken as respiratory specimens (NP) and one CSF). This sampling is what is normally expected. Specimens were taken from the following body sites (and presented as # positive/# specimens) are described below. {10} Specimen Sources | Source | Total specimens | Unknown +/Total | Genital +/Total | Penis +/Total | Vaginal +/Total | Cervical +/Total | Rectal +/Total | Perineum** +/Total | Eyeld +/Total | Face +/Total | Mouth* +/Total | Skin† +/Total | NP+/Total | CSF/Brain +/Total | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | Site 1 | 99 | 0/8 | 0/1 | 0/0 | 0/0 | 0/0 | 0/1 | 0/11 | 0/1 | 4/14 | 0/2 | 17/61 | 0/0 | 0/0 | | Site 2 | 35 | 0/0 | 0/0 | 1/2 | 0/0 | 0/0 | 0/0 | 1/3 | 0/0 | 0/2 | 0/0 | 9/27 | 0/1 | 0/0 | | Site 3 | 120 | 2/51 | 0/6 | 0/1 | 0/9 | 0/1 | 0/0 | 0/3 | 0/0 | 1/9 | 0/5 | 4/33 | 1/1 | 0/1 | | *mouth: mouth, lip, tongue, gum, throat**perineum: groin, buttock, gluteal, coccyx, sacral, pubic, perianal†skin: skin lesion, skin, finger, wrist, chest, axilla, abdomen, thigh, blister | | | | | | | | | | | | | | | Demographics by age and gender for the specimens that were tested at the 3 Study Sites are tabulated below. Of the specimens evaluated in these studies (which had been submitted to the laboratories as swabs taken from lesions for both HSV and VZV testing), a large proportion were from patients between the ages of 18 and 40. Prevalence of VZV within the population tested was quite low (due in part to varicella vaccination programs). The patient demographics are listed below. Demographics by Age and Gender | | Site 1 Values are # pos / Total | | Site 2 Values are # pos / Total | | | Site 3 Values are # pos / Total | | | --- | --- | --- | --- | --- | --- | --- | --- | | Age | F | M | F | M | Gender not reported | F | M | | TOTALS | 63 | 36 | 10 | 10 | 12 | 80 | 40 | | <2y | 0/1 | 0/4 | 0 | 0/2 | 0 | 0 | 0/1 | | 2y to 10y | 0 | 0/1 | 0/1 | 0/1 | 0 | 1/3 | 0/2 | | 10y to18y | 1/6 | 1/3 | 1/1 | 1/1 | 0 | 0/4 | 0/3 | | 18y to 40y | 0/18 | 1/3 | 0/1 | 0/1 | 0 | 0/39 | 0/13 | | >40y | 11/38 | 7/24 | 3/6 | 4/5 | 0 | 2/33 | 5/21 | | Age not reported | 0/0 | 1/1 | 0/1 | 0 | 1/12 | 1/1 | 0 | N. Proposed Labeling: The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10. O. Conclusion: The submitted information in this premarket notification is complete and supports a substantial equivalence decision.