ID-FISH Plasmodium Genus Test Kit, ID-FISH Plasmodium falciparum and P. vivax Combo Test Kit

{0}------------------------------------------------ ## EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR ID-FISH Plasmodium Genus Test Kit ID-FISH Plasmodium falciparum and P. vivax Combo Test Kit DECISION SUMMARY #### A. DEN Number: DEN160025 ### B. Purpose for Submission: De novo request for evaluation of automatic class III designation for the ID-FISH Plasmodium Genus Test Kit and the ID-FISH Plasmodium falciparum and P. vivax Combo Test Kit ### C. Measurands: Ribosomal RNA sequences of Plasmodium falciparum, Plasmodium vivax, and other Plasmodium species ## D. Type of Test: Fluorescence In Situ Hybridization (FISH) assay using fluorescently labeled DNA probes ## E. Applicant: ID-FISH Technology, Inc. ## F. Proprietary and Established Names: ID-FISH Plasmodium Genus Test Kit (PlasG) ID-FISH Plasmodium falciparum and P. vivax Combo Test Kit (PlasFV) ## G. Regulatory Information: - 1. Regulation section: 21 CFR 866.3367 - 2. Classification: Class II (Special Controls) {1}------------------------------------------------ ## 3. Product code(s): PYN - 4. Panel: 83- Microbiology # H. Indications for Use: - 1. Indications for use: ID-FISH Plasmodium Genus Test Kit (PlasG) and ID-FISH Plasmodium falciparum and P. vivax Combo Test Kit (PlasFV) are intended for in vitro diagnostic use in the clinical laboratory for detection of Plasmodium species in human venous whole blood (EDTA) samples from patients suspected of Plasmodium infection. The test kits are intended to aid in the diagnosis of malaria and to aid in the differential diagnosis of P. falciparum and P. vivax infection. The test kits should be used only on samples from patients with a clinical history, signs and symptoms consistent with malaria, and are not intended as a screen for asymptomatic patients. The ID-FISH Plasmodium Genus Test Kit is a qualitative test for detection of malaria parasites in blood smears. Positive results should be supplemented with the Plasmodium species specific test kit, ID-FISH Plasmodium falciparum and P. vivax Combo Test Kit for identification and differentiation of Plasmodium falciparum and Plasmodium vivax. The results of these test kits should be used in conjunction with other diagnostic test results. Clinical performance has not been established for P. ovale, P. malariae, or P. knowlesi. - 2. Special conditions for use statement(s): For in vitro diagnostic use only For prescription use only For professional use only The product has not been validated with specimens other than venous whole blood (EDTA) # 3. Special instrument requirements: Fluorescence microscope equipped with the following: - Mercury arc lamp or LED light powered illumination - Dual band filter set compatible with ID-FISH hybridization probes ■ - 100x oil objective # I. Device Description: The ID-FISH Plasmodium Genus Test Kit (PlasG) and ID-FISH Plasmodium falciparum and {2}------------------------------------------------ P. vivax Combo Test Kit (PlasFV) are fluorescence in situ hybridization (FISH) assays to detect Plasmodium spp. or P. falciparum or P. vivax parasites in thin film blood smears prepared from EDTA venous whole blood samples. Materials provided: ID-FISH Plasmodium Genus Test Kit - Plasmodium Genus Hyb A - Plasmodium Hyb B ● - 5x Plasmodium Wash Buffer - 10x Plasmodium Rinse Buffer ● - Plasmodium Counterstain ● ID-FISH Plasmodium falciparum and P. vivax Combo Test Kit - Plasmodium falciparum / P. vivax Hyb A ● - Plasmodium Hyb B - 5x Plasmodium Wash Buffer ● - 10x Plasmodium Rinse Buffer - Plasmodium Counterstain ● Materials not provided, but available for separate purchase: - SPR Smear Preparation Reagent . - . Dual Band Filter Set specific to ID-FISH hybridization probes # Materials required but not provided: - Fluorescence microscope with 100x oil objective . - 37±1° C Incubator ● - Negative Control Smears (made with non-malarial EDTA blood less than four days old and SPR) - Positive Control Smears (made with EDTA blood infected with P.f. or P.v. parasites less ● than four days old with SPR) # J. Standard/Guidance Document Referenced (if applicable): Not applicable # K. Test Principle: Fluorescent in situ hybridization (FISH) is a microscopic technique in which a DNA probe is labeled with a fluorescent dye and then hybridized on a slide with a target region of nucleic acid of the pathogen in a clinical sample. Venous whole blood from a patient (EDTA preserved) suspected of malaria is mixed with a fixative (smear preparation reagent), smeared onto a glass slide in a thin film, air-dried, and preserved with methanol. The smear {3}------------------------------------------------ is then hybridized with a fluorescently-labeled DNA probe complementary to the rRNA of Plasmodium genus, P. falciparum, or P. vivax, depending on the probe used. Plasmodium parasites are visible in the sample when viewed with a fluorescence microscope equipped with the appropriate filters, and non-Plasmodium organisms do not produce a signal under the filters. Plasmodium species, if present, will appear green with the Plasmodium genus probe in the PlasG test kit. P. falciparum will appear green with the P. falciparum probe, and P. vivax will appear orange with the P. vivax probe in the PlasFV test kit. Ribosomal RNA is present in the cytoplasm of the parasite and is visible within the host red blood cell so that the entire parasite will give a fluorescent signal. Both sexual forms of Plasmodium spp., P. falciparum and P. vivax parasites are labelled by the respective PlasG and PlasFV test kit probes. Samples from patients who are receiving anti-malarial drug treatment may have negative FISH results, while Giemsa microscopy and PCR may remain positive for longer periods of time after treatment. ## L. Performance Characteristics: ### 1. Analytical performance: - a. Reproducibility A study was conducted to evaluate the reproducibility of the ID-FISH Plasmodium Genus Test Kit (PlasG) and the ID-FISH Plasmodium falciparum and P. vivax Combo Test Kit (PlasFV). A panel of five samples was contrived in human EDTA whole blood at the following parasite concentrations: - Moderate positive P. falciparum 480 parasites/uL ● - Moderate positive P. vivax 440 parasites/uL ● - Low positive P. falciparum 156 parasites/uL ● - Low positive P. vivax 160 parasites/uL ● - Negative 0 parasites/uL . One hundred and eighty (180) replicate slides containing two smears per slide were prepared from each sample at two separate sites (900 slides total). The randomized and blinded panel was distributed to three sites (300 slides per site) and tested over five non-consecutive days, with two operators per site performing two runs per day. For each run, each blinded panel member was tested in triplicate with both the PlasG and PlasFV test kits using two smears per slide (3 replicates x 2 operators x 2 runs x 5 days x 3 sites = 180 observations per kit). Of the 900 slides, three smears (one negative smear and two low positive P. falciparum smears) fell off the slides at one site and so could not be read (3/900 = 0.3%). These smears were excluded from the analysis. For the PlasG test kit, one negative smear gave a false positive result in one run for a negative agreement of 99.4% (178/179). There was 100% agreement with the expected result with the PlasG test kit for all other panel members across runs, days, and sites. See Table 1. For the PlasFV test kit, one false positive result was observed for one negative smear (note: the same negative smear that also gave a false positive result with the PlasG test kit) for a negative agreement of 99.4% (178/179). One low positive P. falciparum {4}------------------------------------------------ sample produced a false negative result in one run for an agreement of 99.4% (177/178). There was 100% agreement with the expected P. falciparum, P. vivax, or negative results with the PlasFV test kit for all other panel members across operators, runs, days, and sites. See Table 2. | Sample | Agreement with expected result (%) | | | | | | Overall | 95%<br>Confidence<br>Interval | |-----------|------------------------------------|----------------|----------------|----------------|----------------|-------------------|---------------------|-------------------------------| | | Site 1 | | Site 2 | | Site 3 | | | | | | Op 1 | Op 2 | Op 1 | Op 2 | Op 1 | Op 2 | | | | Negative | 30/30<br>(100) | 30/30<br>(100) | 30/30<br>(100) | 30/30<br>(100) | 30/30<br>(100) | 28a/29b<br>(96.6) | 178a/179b<br>(99.4) | 96.9 - 99.9% | | Low P.f. | 30/30<br>(100) | 30/30<br>(100) | 30/30<br>(100) | 30/30<br>(100) | 30/30<br>(100) | 28/28b<br>(100) | 178/178b<br>(100) | 97.9 – 100% | | Mod. P.f. | 30/30<br>(100) | 30/30<br>(100) | 30/30<br>(100) | 30/30<br>(100) | 30/30<br>(100) | 30/30<br>(100) | 180/180<br>(100) | 97.9-100% | | Low P.v. | 30/30<br>(100) | 30/30<br>(100) | 30/30<br>(100) | 30/30<br>(100) | 30/30<br>(100) | 30/30<br>(100) | 180/180<br>(100) | 97.9-100% | | Mod. P.v. | 30/30<br>(100) | 30/30<br>(100) | 30/30<br>(100) | 30/30<br>(100) | 30/30<br>(100) | 30/30<br>(100) | 180/180<br>(100) | 97.9-100% | Table 1. PlasG test kit reproducibility a One negative smear gave a positive result for Operator 2 at Site 3. b Smear(s) fell off. Table 2. PlasFV test kit reproducibility | | Agreement with expected result (%) | | | | | | | | |-----------|------------------------------------|----------------|----------------|----------------|----------------|-------------------|---------------------|------------------------| | | Site 1 | | Site 2 | | Site 3 | | | 95% | | Sample | Op 1 | Op 2 | Op 1 | Op 2 | Op 1 | Op 2 | Overall | Confidence<br>Interval | | Negative | 30/30<br>(100) | 30/30<br>(100) | 30/30<br>(100) | 30/30<br>(100) | 30/30<br>(100) | 28a/29b<br>(96.6) | 178a/179b<br>(99.4) | 96.9 - 99.9% | | Low P.f. | 30/30<br>(100) | 30/30<br>(100) | 30/30<br>(100) | 30/30<br>(100) | 30/30<br>(100) | 27c/28b<br>(96.4) | 177c/178b<br>(99.4) | 96.9 - 99.9% | | Mod. P.f. | 30/30<br>(100) | 30/30<br>(100) | 30/30<br>(100) | 30/30<br>(100) | 30/30<br>(100) | 30/30<br>(100) | 180/180<br>(100) | 97.9 - 100% | | Low P.v. | 30/30<br>(100) | 30/30<br>(100) | 30/30<br>(100) | 30/30<br>(100) | 30/30<br>(100) | 30/30<br>(100) | 180/180<br>(100) | 97.9 - 100% | | Mod. P.v | 30/30<br>(100) | 30/30<br>(100) | 30/30<br>(100) | 30/30<br>(100) | 30/30<br>(100) | 30/30<br>(100) | 180/180<br>(100) | 97.9 - 100% | a One negative smear gave a false positive result for Operator 2 at Site 3. b Smear(s) fell off. C One low positive P.f. smear gave a false negative result for Operator 2 at Site 3. - b. Linearity/assay Reportable Range: #### Not Applicable - c. Traceability, Stability, Expected Values (controls, calibrators, or methods): #### Recommended External Controls: Control material should be tested in accordance with the guidelines or requirements of local, state, and/or federal regulations or accrediting organizations. To monitor the {5}------------------------------------------------ assay, reagent performance and day-to-day variation, positive controls for P. falciparum or P. vivax along with a negative control (non-malarial blood) must be tested with each run. Negative controls can be prepared from EDTA whole blood collected from normal subjects. Positive slides can be prepared from previously positive patient blood samples. Control slides should be prepared in the same manner as patient samples and they are stable for five years if stored in a dry place at room temperature. During the clinical testing conducted at three sites, external positive and negative FISH controls were tested for each run as per kit instructions, and no control failures were observed for the 61 days of non-consecutive clinical testing across all sites. - d. Detection Limit: The analytical limit of detection (LoD) was estimated for the PlasG and PlasFV test kits using well defined clinical samples serially diluted in EDTA whole blood. A P. falciparum positive sample with a neat parasitemia of 18,375 parasites per microliter and a P. vivax positive sample with a neat parasitemia of 5,040 parasites per microliter were tested. The P. falciparum sample was serially diluted as follows: 1:16, 1:32, 1:64, 1:128, and 1:256. The P. vivax sample was serially diluted as follows: 1:5, 1:10, 1:20, 1:40, 1:80, 1:160, and 1:320. Triplicate smears were prepared from each dilution and tested with both kits, and the highest dilution (i.e. lowest parasite concentration) which produced a positive result for all three replicates was considered the preliminary LoD. The LoD was confirmed by testing an additional 20 smears prepared from blood containing parasites at this concentration to demonstrate a detection rate of greater than or equal to 95%. The LoD for each kit is summarized in Table 3. Table 3. Limit of detection (parasites/uL) | | P.<br>falciparum | P. vivax | |-------------|------------------|----------| | PlasG FISH | 143 | 126 | | PlasFV FISH | 143 | 126 | - e. Analytical Reactivity (Inclusivity): The inclusivity of the PlasG and PlasFV test kits was evaluated with an additional 11 blood samples positive for Plasmodium species. Ten human clinical blood samples from different geographic regions were tested including four P. falciparum, two P. vivax, two P. ovale, and two P. malariae positive samples. One monkey blood P. knowlesi positive sample was also tested. All Plasmodium positive samples were detected by the PlasG test kit. All P. falciparum and P. vivax positive samples were correctly identified by the respective probes in the PlasFV test kit. - Analytical Specificity: f. #### Cross reactivity The cross reactivity of the PlasG and PlasFV test kits was evaluated with a panel of 29 pathogenic bacteria, parasites, and viruses that may be found in human blood {6}------------------------------------------------ samples. Microorganisms were spiked into whole blood samples and then blood smear slides were prepared according to the kit instructions. Except as noted, all parasites and bacteria were tested at concentrations greater than 10° organisms per milliliter and viruses were tested at greater than 10° PFU or copies per milliliter. For the PlasFV test kit. off-target Plasmodium species were also evaluated (e.g., non-P. vivax species to evaluate the P. vivax probe cross reactivity). See Table 4. No offtarget organism produced a positive result with PlasG or PlasFV test kits. Table 4. Cross reactivity panel | Microorganisms | |----------------------------------| | Anaplasma phagocytophilum | | Babesia duncani | | Babesia microti | | Bartonella henselae | | Borrelia burgdorferi | | Bwamba Fever Virus | | Chikungunya virus | | Cytomegalovirus (CMV) | | Dengue virus | | Ehrlichia chaffeensis | | Epstein-Barr virus (EBV) | | Hepatitis B | | HIV-1a | | Influenza A | | Influenza B | | Leishmania major (amastigotes) | | Leishmania major (promastigotes) | | Leptospira interrogans | | Rickettsia rickettsii | | Rickettsia typhi | | Trypanosoma cruzi | | West Nile virus | | Yellow Fever virus | | Plasmodium berghei a,b | | Plasmodium falciparum a,b | | Plasmodium malariae a,b | | Plasmodium knowlesi a,b | | Plasmodium ovale a,b | | Plasmodium vivax a,b | a Tested at > 104 organisms/mL 6 All on-target Plasmodium species were detected with the expected PlasG and PlasFV test kit probes. Additionally, cross reactivity was evaluated with a panel of 76 blood samples from patients with non-malarial medical conditions that were processed as blood smears according to the kit instructions. These included 42 blood samples from patients with positive antibody test results for one or more of the following tick-borne diseases: B. burgdorferi, Babesia spp, B. henselae, A. phagocytophilum, E. chaffeensis, and Rickettsig spp .; 20 samples from patients with positive antimuclear antibody test results; four samples from patients with hepatitis B infection; and ten blood samples spiked with plasma from ten patients with HIV 1 infection. No false positive results were observed with the PlasG or PlasFV test kits. {7}------------------------------------------------ #### Interfering Substances The performances of PlasG and PlasFV test kits were evaluated in the presence of potentially interfering exogenous and endogenous substances that may occur in blood. Human whole blood samples spiked with P. falciparum, P. vivax, or unspiked controls were prepared as smears according to the kit instructions and tested in the presence of potential interferents at the concentrations shown in Tables 5 and 6. Each Plasmodium positive sample was detected by the expected PlasG and PlasFV test kit probes in the presence of the spiked substances. No false positive results were observed in the uninfected control blood samples in the presence of the spiked substances. | Substance | Concentration | |---------------|---------------| | Triglycerides | 37 mmol/L | | Hemoglobin | 2 g/L | | Bilirubin | 342 µmol/L | | Serum albumin | 60 g/L | #### Table 5. Endogenous potential interfering substances | | Table 6. Exogenous potential interfering substances | | | | |--|-----------------------------------------------------|--|--|--| |--|-----------------------------------------------------|--|--|--| | Substance | Concentration | |---------------|---------------| | Acetaminophen | 1 mg/mL | | Aspirin | 1 mg/mL | | Ibuprofen | 1 mg/mL | | Artesunate | 1 mg/mL | | Chloroquine | 1 mg/mL | | Doxycycline | 1 mg/mL | | Primaquine | 1 mg/mL | | Quinine | 1 mg/mL | | Sulfadoxine | 1 mg/mL | | Amoxicillin | 100 µg/mL | | Cephalexin | 100 µg/mL | | Ciprofloxacin | 100 µg/mL | | Erythromycin | 100 ug/mL | - g. Assay Cut-off: n/a {8}------------------------------------------------ #### h. Specimen Stability The specimen collection and handling instructions recommend that blood smear slides for the PlasG and PlasFV test kits should be prepared and fixed as soon as possible after venous whole blood sample collection into EDTA vacutainer tubes. In the clinical study, blood smears were prepared from leftover EDTA venous whole blood samples that were stored at 15 – 30 °C for less than 24 hours or stored at 4 °C for less than four days. The clinical study results supported the storage of whole blood under these conditions prior to preparing blood smear slides. To evaluate the stability of blood smear slides that have been prepared and fixed according to package insert instructions, a real-time sample stability study was conducted. Multiple slide replicates were prepared from each of four P. falciparum and 10 P. vivax positive blood samples that had different concentrations of parasites as determined by microscopic examination (low, medium, or high parasite concentrations. The slides were prepared and stored with desiccant in the dark at 15 -30 °C. The PlasG and PlasFV test kits were used to stain selected slide replicates every three months for the first year, and once per year thereafter up to five years. All samples produced the expected test kit result at each time point without a noticeable reduction in staining intensity. The results supported the storage of fixed slides for specimens or quality controls for up to five years prior to staining. - Fresh versus Frozen Study i. n/a - 2. Comparison Studies: - a. Clinical Comparison: See section L3 - b. Matrix Equivalence Study n/a - 3. Clinical Studies: The clinical performances of the PlasG and PlasFV test kits were evaluated in a multicenter study conducted in malaria endemic and non-endemic regions. The test kit results were compared to Giemsa thick and thin smear malaria microscopy results that followed a uniform protocol with pre-defined Giemsa adjudication among multiple readers. PCR testing followed by bi-directional sequencing was used for discrepant analysis. {9}------------------------------------------------ #### Sample collection A total of 1067 leftover venous whole blood (EDTA) samples were collected from patients with malaria-like symptoms at different times between 2013 and 2015 (See Table 9). Endemic patient ages ranged from 29 days old to 92 years old, and non-endemic patient ages ranged from two vears old to 92 vears old (See Tables 7 - 8). Of the 917 patient samples enrolled from malaria endemic regions. 300 sequential samples (i.e., all comers meeting inclusion criteria) were collected from a site in Mangalore India, 395 sequential samples were collected from three Kenyan sites that receive samples from western and central Kenya (one site in Kisumu, Kenya and two sites in Nairobi, Kenya), and 222 samples were collected from Iquitos, Peru. The 222 samples from Peru were selectively enrolled (non-sequential) based on Giemsa results as follows: 22 Giemsa positive samples, and an additional 100 Giemsa positive samples with 100 matched Giemsa negative samples. A total of 150 non-endemic symptomatic patient samples were collected from the United States. An additional 21 samples that were collected from Kenya were excluded from analysis as per the study protocol because the patients had received malaria treatment within the six weeks prior to sample collection. | Patient Age group | Kenya | India | Peru | Total<br>Samples<br>Tested | Percent | |-----------------------|-------|-------|------|----------------------------|---------| | 29 days - < 2 years | 8 | 2 | 0 | 10 | 1.1% | | 2 years - < 12 years | 154 | 10 | 22 | 186 | 20.3% | | 12 years - < 18 years | 41 | 18 | 12 | 71 | 7.7% | | 18 - < 21 years | 10 | 20 | 13 | 43 | 4.7% | | 21 years or older | 180 | 248 | 175 | 603 | 65.8% | | Unknown | 2 | 2 | 0 | 4 | 0.4% | | Total | 395 | 300 | 222 | 917 | 100% | Table 7. Patient Age Ranges - Endemic Regions | Table 8. Patient Age Ranges - Non-Endemic Region - US | | | | | | | | |-------------------------------------------------------|--|--|--|--|--|--|--| |-------------------------------------------------------|--|--|--|--|--|--|--| | Patient Age group | Total<br>Samples<br>Tested | Percent | |-----------------------|----------------------------|---------| | 29 days - < 2 years | 0 | 0% | | 2 years - < 12 years | 5 | 3% | | 12 years - < 18 years | 6 | 4% | | 18 - < 21 years | 4 | 3% | | 21 years or older | 134 | 89% | | Unknown | 1 | 1% | | Total | 150 | 100% | {10}------------------------------------------------ | Endemic/<br>Non-Endemic | Country | Study<br>enrollment | #<br>Specimens | |-------------------------|---------------|---------------------|----------------| | Non-Endemic | United States | All comers | 150 | | Endemic | India | All comers | 300 | | | Kenya | All comers | 395 | | | Peru | Selective | 222 | | Total | | | 1067 | Table 9. Sample Collection Totals ### Sample testing Giemsa thick and thin smear malaria microscopy testing was conducted at each collection site using a uniform protocol and pre-defined, expert adjudication by up to three microscopists. Venous whole blood smears prepared as per the ID-FISH protocol were evaluated by the PlasG and PlasFV test kits at three testing sites: one site in Mangalore, India (testing the Indian samples, n=300), one site in Nairobi, Kenya (testing the majority of the Kenyan samples, n=299), and the sponsor internal site in Palo Alto, CA (testing the US samples, n=150, the Peruvian samples, n=222, and the remaining Kenyan samples not previously tested, n=96). All samples were blinded and coded prior to testing, and all FISH testing and comparator method testing procedures were conducted by independent technicians. External positive and negative FISH controls were tested for each run, and no control failures were observed for the 61 days of non-consecutive clinical testing across all sites. The results are summarized below. ## Non-endemic region sample results All 150 non-endemic symptomatic patient samples from the US had negative Giemsa microscopy results, and all samples produced negative results with the PlasG and PlasFV test kits. The specificity for each test kit in this population in comparison to Giemsa microscopy was 100% (150/150) 95% CI 95.0 - 99.2%. Malaria endemic region sample results (all comers enrollment India and Kenva) Results from sequential (all-comers) samples from India and Kenya are presented combined (n=695, Tables 10 - 12), and also separately by collection source (India n=300, Tables 13 - 15 and Kenya n=395. Tables 16 - 18). The Kenvan sample results from different collection sites and testing sites are shown combined as the test performances were similar across sites. ## PlasG FISH test kit In comparison to expert adjudicated Giemsa microscopy for detecting any Plasmodium sp., the PlasG test kit had a sensitivity point estimate greater than 95% with the lower bound of the two-sided 95% CI greater than 90% with the samples collected from India and Kenya. The PlasG test kit specificity point estimate was 92.6% for these endemic samples; however, the results of PCR/sequencing demonstrating the presence of parasite DNA in 20 of the 27 false positive samples indicate that the specificity is likely to be higher (Table 10). {11}------------------------------------------------ #### PlasFV FISH test kit – P. falciparum In comparison to expert adjudicated Giemsa microscopy for detecting and identifying P. falciparum with the samples collected from India and Kenya, the sensitivity of the PlasFV test kit was 97.4%(152/156) 95% CI 93.6% - 99.0%. The specificity for P. falciparum in this data set was 96.1%; however, PCR/sequencing results demonstrating the presence of P. falciparum DNA in 17 of the 20 false positive samples indicate that the specificity is likely to be higher (Table 11). #### PlasFV FISH test kit - P. vivax No P. vivax positive samples were collected from Kenya (Table 18). With the samples collected from India, the sensitivity of the PlasFV test kit for identifying P. vivax was 91.2% (156/171) 95% CI 86.0 - 94.6% in comparison to adjudicated Giemsa microscopy (Table 15). When results with prospective samples from India and Kenya are combined, the specificity of the PlasFV test kit for P. vivax was 98.3% (515/524) 95% CI 96.8 -99.1%; however, PCR/sequencing results demonstrating the presence of P. vivax DNA in 6 of the 9 false positive samples indicate that the specificity is likely to be higher (Table 12). {12}------------------------------------------------ ### Combined Sample Results - India and Kenya | | | Giemsa Microscopy | | | |------------|------------------|-------------------|-------|-------------| | | | Pos | Neg | Grand Total | | PlasG FISH | Pos | 314b | 27a | 341 | | | Neg | 14 | 340 | 354 | | | Grand Total | 328 | 367 | 695 | | Sens 95.7% | (314/328) 95% CI | 93.0% | 97.4% | | | Spec 92.6% | (340/367) 95% CI | 89.5% | 94.9% | | Table 10. PlasG test kit vs. Giemsa microscopy - All Plasmodium sp :: Combined Indian and Kenyan Sample Results a Of the 27 samples with Giemsa negative and PlasG test kit positive results, 20 samples were positive for Plasmodium species DNA by PCR/sequencing: 13 P. falciparum DNA positive, 6 P. vivax DNA positive, and 1 P. malariae DNA positive. of Of the 314 concordant Giemsa positive and PlasG test kit positive results, 2 samples were positive for P. ovale DNA and 1 sample was positive for P. malariae DNA by PCR/sequencing. Table 11. PlasFV test kit vs. Giemsa Microscopy - P. falciparum: Combined Indian and Kenyan Sample Results | | | Giemsa Microscopy | | | |-------------|-----------------|-------------------|-----------------|-------------| | | | <i>P. f.</i> Pos | <i>P.f.</i> Neg | Grand Total | | PlasFV FISH | <i>P.f.</i> Pos | 152 | 21a | 173 | | | <i>P.f.</i> Neg | 4 | 518 | 522 | | | Grand Total | 156 | 539 | 695 | | Sens | 97.4% | (152/156) | 95% CI | 93.6% | 99.0% | |------|-------|-----------|--------|-------|-------| | Spec | 96.1% | (518/539) | 95% CI | 94.1% | 97.4% | a Of the 21 samples negative for P. falciparum by Giemsa and positive for P. falciparum by the PlasFV test kit, 17 samples were positive for P. falciparum DNA by PCR/sequencing. | | Table 12. PlasFV test kit vs. Giemsa Microscopy - P. vivax: Combined Indian and | | |-----------------------|---------------------------------------------------------------------------------|--| | Kenyan Sample Results | | | | | Carrely Company Component Company Comments of Children | | | | | Giemsa Microscopy | | | |-------------|-----------------|-------------------|-----------------|-------------| | | | <i>P.v.</i> Pos | <i>P.v.</i> Neg | Grand Total | | PlasFV FISH | <i>P.v.</i> Pos | 156 | 9a | 165 | | | <i>P.v.</i> Neg | 15 | 515 | 530 | | | Grand Total | 171 | 524 | 695 | | Sens | 91.2% | (156/171) | 95% CI | 86.0% | 94.6% | |------|-------|-----------|--------|-------|-------| | Spec | 98.3% | (515/524) | 95% CI | 96.8% | 99.1% | a Of the 9 samples negative for P. vivax by Giemsa negative and positive for P. vivax by the PlasFV test kit, 6 samples were positive for P. vivax DNA by PCR/sequencing. One sample that was positive for P. falciparum by Giemsa and positive for P. falciparum DNA by PCR/sequencing is included in this count of samples negative for P. vivax by Giemsa. {13}------------------------------------------------ #### Sample Results - India | Table 13. PlasG test kit vs. Giemsa microscopy - All Plasmodium sp .: Indian Sample | | | |-------------------------------------------------------------------------------------|--|--| | Results | | | | | Giemsa Microscopy | | | | |------------|-------------------|-----|-------------|-----| | | Pos | Neg | Grand Total | | | PlasG FISH | Pos | 179 | 9a | 188 | | | Neg | 11 | 101 | 112 | | | Grand Total | 190 | 110 | 300 | | Sens | 94.2% | (179/190) | 95% CI | 89.9% 96.7% | |------|-------|-----------|--------|-------------| | Spec | 91.8% | (101/110) | 95% CI | 85.2% 95.6% | a Of the 9 samples with Giemsa negative and FISH positive results, 7 samples were positive for Plasmodium species DNA by PCR/sequencing: 6 samples positive for P. vivax DNA and 1 sample positive for P. falciparum DNA. Table 14. PlasFV test kit vs. Giemsa Microscopy - P. falciparum: Indian Sample Results | | | Giemsa Microscopy | | | |-------------|-----------------|-------------------|-----------------|-------------| | | | <i>P. f.</i> Pos | <i>P.f.</i> Neg | Grand Total | | PlasFV FISH | <i>P.f.</i> Pos | 19 | 5a | 24 | | | <i>P.f.</i> Neg | 0 | 276 | 276 | | | Grand Total | 19 | 281 | 300 | | Sens | 100% | (19/19) | 95% CI | 83.2% | 100.% | |------|-------|-----------|--------|-------|-------| | Spec | 98.2% | (276/281) | 95% CI | 95.9% | 99.2% | a All 5 samples with Giemsa negative and FISH positive results were positive for P. falciparum DNA by PCR/sequencing. | | | Giemsa Microscopy | | | |-------------|-------------|-------------------|----------|-------------| | | | P.v. Pos | P.v. Neg | Grand Total | | PlasFV FISH | P.v. Pos | 156 | 9a | 165 | | | P.v. Neg | 15b | 120 | 135 | | | Grand Total | 171 | 129 | 300 | Table 15. PlasFV test kit vs. Giemsa Microscopy - P. vivax: Indian Sample Results | Sens | 91.2% | (156/171) | 95% CI | 86.0% | 94.6% | |------|-------|-----------|--------|-------|-------| | Spec | 93.0% | (120/129) | 95% CI | 87.3% | 96.3% | a Of the 9 samples Giemsa negative and FISH positive for P. vivax, 6 samples were positive for P. vivax DNA by PCR/sequencing. One sample that was positive for P. falciparum DNA by PCR/sequencing and Giemsa positive for P. falciparum is included in the count of samples negative for P. vivax by Giemsa. b Of the 15 samples that were positive for P. vivax by Giemsa and negative for P. vivax by the PlasFV test kit, 4 samples were positive for Plasmodium sp. by the PlasG test kit. All 4 samples were positive for P. falciparum DNA by PCR/sequencing; however, dual infection with P. vivax could not be ruled out by this method. 3 of the 4 sample were positive for P. falciparum by the PlasFV test kit {14}------------------------------------------------ #### Sample Results - Kenya | | | Table 16. PlasG Kit vs. Giemsa microscopy - All Plasmodium sp .: Combined Kenyan | |----------------|--|----------------------------------------------------------------------------------| | Sample Results | | | | | | Giemsa Microscopy | | | |------------|-------------|-------------------|-----|-------------| | | | Pos | Neg | Grand Total | | PlasG FISH | Pos | 135b | 19a | 154 | | | Neg | 3 | 238 | 241 | | | Grand Total | 138 | 257 | 395 | | Sens | 97.8% | (135/138) | 95% CI | 93.8% | 99.3% | |------|-------|-----------|--------|-------|-------| | Spec | 92.6% | (238/257) | 95% CI | 88.7% | 95.2% | a Of the 19 samples with Giemsa negative and positive PlasG test kit results, 13 samples were positive for Plasmodium species DNA by PCR/sequencing: 12 samples positive for P. falciparum DNA and 1 sample positive for P. malariae DNA. b Of the 135 Giemsa positive and FISH positive results, 2 samples were positive for P. ovale DNA and 1 sample was positive for P. malariae DNA by PCR/sequencing. Table 17. PlasFV test kit vs. Giemsa Microscopy - P. falciparum: Combined Kenyan Sample Results | | | Giemsa Microscopy | | | |-------------|-----------------|-------------------|-----------------|-------------| | | | <i>P. f.</i> Pos | <i>P.f.</i> Neg | Grand Total | | PlasFV FISH | <i>P.f.</i> Pos | 133 | 16a | 149 | | | <i>P.f.</i> Neg | 4b | 242 | 246 | | | Grand Total | 137 | 258 | 395 | | Sens | 97.1% | (133/137) 95% CI | 92.7% | 98.9% | |------|-------|------------------|-------|-------| | Spec | 93.8% | (242/258) 95% CI | 90.2% | 96.2% | a Of the 16 samples with Giemsa negative and FISH positive results, 12 samples were positive for P. falciparum DNA by PCR/sequencing. b Of the 4 samples positive for P. falciparum by Giemsa and negative for P. falciparum by the PlasFV test kit, 1 sample was positive for Plasmodium sp. by the PlasG test kit. This sample was positive for P. ovale DNA by PCR/sequencing. Table 18. PlasFV test kit vs. Giemsa Microscopy - P. vivax: Combined Kenyan Sample Results | | | Giemsa Microscopy | | | |-------------|-------------|-------------------|----------|-------------| | | | P.v. Pos | P.v. Neg | Grand Total | | PlasFV FISH | P.v. Pos | 0 | 0 | 0 | | | P.v. Neg | 0 | 395 | 395 | | | Grand Total | 0 | 395 | 395 | | Sens | n/a | | | | | |------|------|-----------|--------|-------|------| | Spec | 100% | (395/395) | 95% CI | 99.0% | 100% | {15}------------------------------------------------ #### Malaria endemic region sample results (selective enrollment Peru) The Peru sample results are presented separately as positive percent agreement and negative percent agreement (PPA, NPA) with adjudicated Giemsa microscopy because this sample set was selectively enrolled based on Giemsa results (Peru n=222, Tables 19 -21). #### PlasG FISH test kit The PlasG test kit had a PPA of 95.9% (117/122) 95% CI 90.8 - 98.2% and a NPA of 100% (100/100) 95% CI 96.3 - 100% in comparison to Giemsa results for detecting any Plasmodium sp. with the samples collected from Peru (Table 19). #### PlasFV FISH test kit – P. falciparum The PlasFV test kit had a PPA of 100% (5/5) 95% CI 56.6 - 100% and a NPA of 100% (217/217) 95% CI 98.3 - 100% for P. falciparum in comparison to Giemsa results with the samples collected from Peru (Table 20). #### PlasFV FISH test kit – P. vivax The PlasFV test kit had a PPA of 90.6% (106/117) 95% CI 84.0 - 94.7% and a NPA of 100% (105/105) 95% CI 96.5 -100% for P. vivax in comparison to Giemsa results with the samples collected from Peru. Of the 11 samples reported as Giemsa positive and PlasFV test kit negative for P. vivax, seven samples were positive for Plasmodium sp. by the PlasG test kit (Table 21) {16}------------------------------------------------ ## Sample Results - Peru Table 19. PlasG test kit vs. Giemsa microscopy - All Plasmodium sp .: Peruvian Sample Results | | Giemsa Microscopy | | | | |------------|-------------------|-----|-------------|-----| | | Pos | Neg | Grand Total | | | PlasG FISH | Pos | 117 | 0 | 117 | | | Neg | 5 | 100 | 105 | | | Grand Total | 122 | 100 | 222 | | | PPA | 95.9% | (117/122) | 95% CI | 90.8% | 98.2% | |--|-----|-------|-----------|--------|-------|-------| | | NPA | 100% | (100/100) | 95% CI | 96.3% | 100% | Table 20. PlasFV test kit vs. Giemsa Microscopy - P. falciparum: Peruvian Sample Results | | | Giemsa Microscopy | | | |-------------|-----------------|-------------------|-----------------|-------------| | | | <i>P. f.</i> Pos | <i>P.f.</i> Neg | Grand Total | | PlasFV FISH | <i>P.f.</i> Pos | 5 | 0 | 5 | | | <i>P.f.</i> Neg | 0 | 217 | 217 | | | Grand Total | 5 | 217 | 222 | | PPA | 100% | (5/5) | 95% CI | 56.6% | 100% | |-----|------|-----------|--------|-------|------| | NPA | 100% | (217/217) | 95% CI | 98.3% | 100% | Table 21. PlasFV test kit vs. Giemsa Microscopy - P. vivax: Peruvian Sample Results | | Giemsa Microscopy | | | | |-------------|-------------------|----------|----------|-------------| | PlasFV FISH | | P.v. Pos | P.v. Neg | Grand Total | | | P.v. Pos | 106 | 0 | 106 | | | P.v. Neg | 11a | 105 | 116 | | | Grand Total | 117 | 105 | 222 | | PPA | 90.6% | (106/117) | 95% CI | 84.0% | 94.7% | |-----|-------|-----------|--------|-------|-------| | NPA | 100% | (105/105) | 95% CI | 96.5% | 100% | a Of the 11 samples reported as Giemsa positive and FISH negative for P. vivax by the PlasFV test kit, 7 samples were positive for Plasmodium sp. by the PlasG test kit. {17}------------------------------------------------ #### Positive results stratified by parasitemia Kit performance for all positive samples combined is also presented stratified by the Giemsa microscopy quantified parasite level (PlasG FISH Tables 22 - 24, and PlasFV FISH Tables 25 - 26). The sensitivity point estimate for the PlasG test kit for all species combined was 94.7% at parasite concentrations of 101 - 500 parasites per microliter, and 100% at concentrations above 5000 parasites per microliter (Table 22). The sensitivity point estimate of the PlasFV test kit for P. falciparum was 93.3% at parasite concentrations of 101 - 500 parasites per microliter, and 100% at concentrations above 500 parasites per microliter (Table 25). The sensitivity point estimate of the PlasFV test kit for P. vivax was 92.6 -97.5% at parasite concentrations between 1001 - 10,000 parasites per microliter, and 100% at higher concentrations (Table 26). {18}------------------------------------------------ # Combined Results Stratified by Parasitemia - PlasG test kit | Parasites/µl<br>blood | Sensitivity | (PlasG FISH Pos/<br>Giemsa sp. Pos) | 95% CI | | |-----------------------|-------------|-------------------------------------|--------|-------| | >10000 | 100% | (63/63) | 94.3% | 100% | | 5001-10000 | 100% | (99/99) | 96.3% | 100% | | 1001-5000 | 95.8% | (113/118) | 90.5% | 98.2% | | 501-1000 | 98.4% | (62/63) | 91.5% | 99.7% | | 101-500 | 94.7% | (71/75) | 87.1% | 97.9% | | <100 | 71.9% | (23/32) | 54.6% | 84.4% | | Total | 95.8% | (431/450) | 93.5% | 97.3% | Table 22. PlasG test kit vs. Giemsa Microscopy for All Plasmodium Species Table 23. Combined Results Stratified by Parasitemia. PlasG test kit vs. Giemsa Microscopy for P. falciparum | Parasites/µl<br>blood | Sensitivity | (PlasG FISH Pos/<br>Giemsa P.f. Pos) | 95% CI | | |-----------------------|-------------|--------------------------------------|--------|-------| | >10000 | 100% | (38/38) | 90.82% | 100% | | 5001-10000 | 100% | (17/17) | 81.6% | 100% | | 1001-5000 | 100% | (37/37) | 90.6% | 100% | | 501-1000 | 100% | (26/26) | 87.1% | 100% | | 101-500 | 96.7% | (29/30) | 83.3% | 99.4% | | <100 | 84.6% | (11/13) | 57.8% | 95.7% | | Total | 98.1% | (158/161) | 94.7% | 99.4% | Table 24. Combined Results Stratified by Parasitemia. PlasG test kit vs. Giemsa Microscopy for P. vivax | Parasites/μl<br>blood | Sensitivity | (PlasG FISH Pos/<br>Giemsa P.v. Pos) | 95% CI | | |-----------------------|-------------|--------------------------------------|--------|-------| | >10000 | 100% | (25/25) | 86.7% | 100% | | 5001-10000 | 100% | (81/81) | 95.5% | 100% | | 1001-5000 | 93.8% | (76/81) | 86.4% | 97.3% | | 501-1000 | 97.3% | (36/37) | 86.2% | 99.5% | | 101-500 | 93.3% | (42/45) | 82.1% | 97.7% | | <100 | 63.2% | (12/19) | 41.0% | 80.9% | | Total | 94.4% | (272/288) | 91.2% | 96.6% | {19}------------------------------------------------ | Parasites/µl<br>blood | Sensitivity | (PlasFV FISH Pos/<br>Giemsa P.f. Pos) | 95% CI | | |-----------------------|-------------|---------------------------------------|--------|-------| | >10000 | 100% | (38/38) | 90.8% | 100% | | 5001-10000 | 100% | (17/17) | 81.6% | 100% | | 1001-5000 | 100% | (37/37) | 90.6% | 100% | | 501-1000 | 100% | (26/26) | 87.1% | 100% | | 101-500 | 93.3% | (28/30) | 78.7% | 98.2% | | <100 | 84.6% | (11/13) | 57.8% | 95.7% | | Total | 97.5% | (157/161) | 93.8% | 99.0% | Table 25. Combined Results Stratified by Parasitemia. PlasFV test kit vs. Giemsa Microscopy for P. falciparum Table 26. Combined Results Stratified by Parasitemia. PlasFV test kit vs. Giemsa Microscopy for P. vivax | Parasites/ul<br>blood | Sensitivity | (PlasFV FISH Pos/<br>Giemsa P.v. Pos) | 95% CI | | |-----------------------|-------------|---------------------------------------|--------|-------| | >10000 | 100% | (25/25) | 86.7% | 100% | | 5001-10000 | 97.5% | (79/81) | 91.4% | 99.3% | | 1001-5000 | 92.6% | (75/81) | 84.8% | 96.6% | | 501-1000 | 83.8% | (31/37)b | 68.9% | 92.4% | | 101-500 | 88.9% | (40/45) | 76.5% | 95.2% | | <100 | 63.2% | (12/19) | 41.0% | 80.9% | | Total | 91.0% | (262/288)ª | 87.1% | 93.8% | a Of the 26 samples overall that were positive for P. vivax by Giemsa and negative for P. vivax by the PlasFV test kit, 11 samples were positive for Plasmodium sp. by the PlasG test kit. b Of the 6 samples in this category that were positive for P. vivax by Giemsa and negative for P. vivax by the PlasFV test kit, 2 samples were positive for P. falciparum DNA by PCR/sequencing. {20}------------------------------------------------ ### 4. Clinical cut-off: n/a. This is a qualitative test. - 5. Expected values/Reference range: n/a ## M. Instrument Name n/a ## N. System Descriptions: - 1. Modes of Operation: n/a - 2. Software: n/a - 3. Specimen Identification: n/a - 4. Specimen Sampling and Handling: n/a - 5. Calibration: n/a - 6. Quality Control: See section L1c. ## O. Other Supportive Instrument Performance Characteristics Data Not Covered in the "Performance Characteristics" Section above: Fluorescence Microscope Illumination Comparison A study was conducted to evaluate whether blood smears stained with PlasG and PlasFV test kit reagents can provide equivalent results when visualized with fluorescence microscopes that use either mercury arc lamp or LED fluorescence illumination. A sample panel consisting of high and low concentration P. falciparum, high and low concentration P. vivax, and negative blood samples was prepared as blood smear slides, and the smears were stained with PlasG and PlasFV test kit reagents. The slides were observed on a microscope with LED {21}------------------------------------------------ illumination and on a microscope with mercury arc lamp illumination. No false positive results were observed. All parasite positive slides produced the expected results with both illumination sources, and no signal bleed-through was observed for either illumination source. All other analytical and clinical samples were conducted with LED illumination. The results support the use of either LED or mercury arc lamp illumination with the PlasG and PlasFV test kits. ## P. Proposed Labeling: The labeling is sufficient and satisfies the requirements of 21 CFR parts 801 and 809, as applicable, as well as the Special Controls for this type of device. - 1. Indications for Use statement (Special Control 5): The Indications for Use statement in the device labeling must include a statement that the device is intended to be used in conjunction with clinical history, signs, symptoms, and the results of other diagnostic testing. - 2. Interpretation of results and acceptance criteria (Special Control 6) A detailed explanation of the interpretation of results and acceptance criteria for any quality control testing must be included in the device's 21 CFR 809.10(b)(9) compliant labeling. - 3. Negative results limitation (Special Control 7) The device labeling must include a limitation that negative results do not preclude the possibility of infection. {22}------------------------------------------------ # Q. Identified Risks and Required Mitigations: | Identified Risks to Health | Identified Mitigations (See Section S below for Special Controls) | |----------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------| | Incorrect identification or lack of identification of a pathogenic microorganism by the device can lead to improper patient management | General Controls and Special Controls (1), (2), (3), (4), (5), (6), and (7) | | Failure to correctly interpret test results | General Controls and Special Controls (5), (6), and (7) | | Failure to correctly operate the instrument | General Controls and Special Controls (5) and (6) | # R. Benefit/Risk Analysis: | Summary | | |--------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| | Summary of<br>the Benefit(s) | The PlasG and PlasFV FISH test kits are the first assays to use fluorescent in situ<br>hybridization to detect and identify malaria nucleic acids directly in blood smears<br>prepared from venous EDTA blood samples. The PlasG and PlasFV FISH test kits can assist microscopists with detecting and<br>identifying malaria parasites, and may be particularly valuable at sites where personnel<br>have less experience or training in interpreting malaria smears. The PlasG and PlasFV FISH test kits assist in distinguishing between Plasmodium<br>falciparum and Plasmodium vivax infections. In comparison to expert adjudicated Giemsa microscopy, the PlasG and PlasFV FISH<br>test kits demonstrated sensitivity greater than 95% for the identification of Plasmodium<br>spp. and P. falciparum infections, and sensitivity greater than 90% for the identification<br>of P. vivax infections. | | Summary of<br>the Risk(s) | False positive results and false negative results are the primary risks associated with<br>use of the PlasG and PlasFV FISH test kits. A false positive result may lead to unnecessary antimalarial therapy, with associated<br>adverse events. A false negative result may result in a delay of antimalarial therapy, with subsequent<br>worsening of infection. A false negative result may also result a missed opportunity to<br>provide antimalarial therapy to eliminate malaria hypnozoites, the dormant liver form<br>of P. vivax, with subsequent relapse of infection. | | Summary of<br>Other<br>Factors | None. | {23}------------------------------------------------ | Conclusions | The probable benefits of the PlasG and PlasFV FISH test kits outweigh the potential risks | |--------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| | Do the<br>probable | in light of the listed special controls and applicable general controls, including design | | benefits | controls. The PlasG and PlasFV FISH test kits are the first assays to use fluorescent in situ | | outweigh the | hybridization to detect and identify malaria nucleic acids in blood smear samples, and are | | probable<br>risks? | likely to benefit patients by assisting with the diagnosis and identification of malaria<br>infections. The high performance observed during the pivotal clinical trials in comparison<br>to adjudicated Giemsa microscopy, and the proposed special controls suggest that errors<br>will be uncommon and will be well mitigated by current laboratory practices, including<br>traditional microscopy and product labelling. The PlasG and PlasFV FISH test kits could<br>provide substantial benefits as an aid in the diagnosis of malaria infections. | ### S. Conclusion: The information provided in this de novo submission is sufficient to classify this device into class II under regulation 21 CFR 866.3367. FDA believes that the stated special controls, and applicable general controls, including design controls, provide reasonable assurance of the safety and effectiveness of the device type. The device is classified under the following: | Product Code: | PYN | |---------------|----------------------------------------------------------------------------------------| | Device Type: | Device to detect and identify microbial nucleic acids by FISH in clinical<br>specimens | | Class: | II (special controls) | | Regulation: | 21 CFR 866.3367 | - (a) Identification. A device to detect and identify microbial nucleic acids by fluorescence in situ hybridization (FISH) in clinical specimens is an in vitro diagnostic device intended for the detection and identification of microbial pathogens in specimens collected from patients with signs and symptoms of infection. The device is intended to aid in the diagnosis of human disease in conjunction with clinical signs and symptoms and other laboratory findings. - (b) Classification. Class II (special controls). A device to detect and identify microbial nucleic acids by FISH in clinical specimens must comply with the following special controls: - 1) Premarket notification submissions must include detailed device description documentation, including the device components, instrument requirements. ancillary reagents required but not provided, and a detailed explanation of the methodology including all pre-analytical methods for processing of specimens, probe sequences, and rationale for probe sequence selection. - 2) Premarket notification submissions must include a detailed description of the fluorophores, signal source, detection mechanism and method of result interpretation. - 3) Premarket notification submissions must include detailed documentation from the following analytical studies: analytical sensitivity (Limit of Detection), {24}------------------------------------------------ inclusivity, reproducibility, interference, cross reactivity, and specimen stability. - 4) Premarket notification submissions must include detailed documentation from a clinical study that includes prospective (sequential) samples. The study, performed on a study population consistent with the intended use population, must compare the device performance to results obtained from appropriate and well-accepted comparator methods. - 5) The 21 CFR 809.10(b)(2) compliant labeling must include a statement that the device is intended to be used in conjunction with clinical history, signs, symptoms, and the results of other diagnostic testing. - 6) The 21 CFR 809.10(b)(9) compliant labeling must include a detailed explanation of the interpretation of results and acceptance criteria for any quality control testing. - 7) The 21 CFR 809.10(b)(5)(ii) compliant labeling must include a limitation that negative results do not preclude the possibility of infection. {25}------------------------------------------------