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.

Device Description

The ID-FISH Plasmodium Genus Test Kit (PlasG) and ID-FISH Plasmodium falciparum and 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.

AI/ML Overview

Here's a breakdown of the acceptance criteria and the study proving the device meets them, based on the provided text:

Acceptance Criteria and Reported Device Performance

Acceptance Criteria Category	Specific Criteria/Metric	Target Performance (Implicit from study results)	Reported Device Performance (PlasG)	Reported Device Performance (PlasFV - P. falciparum)	Reported Device Performance (PlasFV - P. vivax)
Reproducibility	Negative Agreement	High agreement (e.g., >95%)	99.4% (178/179)	99.4% (178/179)	N/A (Kit differentiates species)
	Positive Agreement (Low P.f.)	100%	100% (178/178)	99.4% (177/178)	N/A
	Positive Agreement (Mod P.f.)	100%	100% (180/180)	100% (180/180)	N/A
	Positive Agreement (Low P.v.)	100%	100% (180/180)	N/A	100% (180/180)
	Positive Agreement (Mod P.v.)	100%	100% (180/180)	N/A	100% (180/180)
Limit of Detection (LoD)	Detection Rate	>= 95% at specified concentration	143 parasites/uL (P.f.), 126 parasites/uL (P.v.)	143 parasites/uL (P.f.), 126 parasites/uL (P.v.)	143 parasites/uL (P.f.), 126 parasites/uL (P.v.)
Analytical Reactivity (Inclusivity)	Detection of Plasmodium species	All Plasmodium positive samples detected	All 11 Plasmodium positive samples detected	P. falciparum and P. vivax positive samples correctly identified	P. falciparum and P. vivax positive samples correctly identified
Analytical Specificity (Cross-Reactivity)	No false positive results	0 false positives	No false positives with 29 pathogens/76 non-malarial blood samples	No false positives with 29 pathogens/76 non-malarial blood samples (for respective probes)	No false positives with 29 pathogens/76 non-malarial blood samples (for respective probes)
Interfering Substances	No false positives/negatives	No impact on results	No false positives, expected results for positive samples	No false positives, expected results for positive samples	No false positives, expected results for positive samples
Specimen Stability	Stable for 5 years (fixed slides storage)	No noticeable reduction in staining intensity, expected result	All samples produced expected results	All samples produced expected results	All samples produced expected results
Clinical Performance (Endemic Regions - All Comers)	Sensitivity (Plasmodium spp.)	>95% (lower bound 95% CI >90%)	95.7% (95% CI 93.0% - 97.4%)	N/A	N/A
	Specificity (Plasmodium spp.)	High specificity, PCR confirmation for "false positives"	92.6% (95% CI 89.5% - 94.9%) - improved by PCR	N/A	N/A
	Sensitivity (P. falciparum)	High sensitivity	N/A	97.4% (95% CI 93.6% - 99.0%)	N/A
	Specificity (P. falciparum)	High specificity, PCR confirmation for "false positives"	N/A	96.1% (95% CI 94.1% - 97.4%) - improved by PCR	N/A
	Sensitivity (P. vivax)	High sensitivity	N/A	N/A	91.2% (95% CI 86.0% - 94.6%)
	Specificity (P. vivax)	High specificity, PCR confirmation for "false positives"	N/A	N/A	98.3% (95% CI 96.8% - 99.1%) - improved by PCR
Clinical Performance (Non-Endemic Regions)	Specificity (Plasmodium spp.)	High specificity	100% (150/150)	100% (150/150)	100% (150/150)
Clinical Performance (Low Parasitemia)	Sensitivity (Plasmodium spp. <100/µL)	N/A (but reported)	71.9% (23/32)	N/A	N/A
	Sensitivity (P. falciparum <100/µL)	N/A (but reported)	N/A	84.6% (11/13)	N/A
	Sensitivity (P. vivax <100/µL)	N/A (but reported)	N/A	N/A	63.2% (12/19)

Study Details Proving Device Meets Acceptance Criteria

Sample Size and Data Provenance:
- Reproducibility Test Samples: A panel of 5 contrived samples (1 negative, 2 low positive P.f., 1 moderate positive P.f., 1 low positive P.v., 1 moderate positive P.v.) were used. 180 replicate slides (with 2 smears per slide) were prepared from each sample across two separate sites, totaling 900 slides.
- Clinical Test Set:
  - Total: 1067 leftover venous whole blood (EDTA) samples.
  - Provenance:
    - Endemic Regions: 917 samples from India (300 sequential), Kenya (395 sequential from multiple sites), and Peru (222 selective enrollment). Patients had malaria-like symptoms.
    - Non-Endemic Region: 150 symptomatic patient samples from the United States.
  - Retrospective/Prospective: Samples were generally collected between 2013 and 2015. The collection method for endemic regions involved "sequential samples (i.e. all comers meeting inclusion criteria)" for India and Kenya, which implies a prospective component for real-world representation, although the term "leftover" suggests they were archived for study. Peru samples were "selectively enrolled" which indicates a retrospective design for balancing positive/negative cases. Non-endemic samples were "all comers," implying a prospective or representative collection.
Number of Experts and Qualifications for Ground Truth:
- Giemsa Microscopy (Comparator/Ground Truth): "pre-defined, expert adjudication by up to three microscopists."
- Qualifications: The document does not explicitly state the qualifications (e.g., years of experience, board certification) of these microscopists. However, the term "expert adjudication" implies high proficiency.
Adjudication Method for the Test Set:
- Giemsa Microscopy: "pre-defined, expert adjudication by up to three microscopists." This suggests a consensus-based approach (e.g., majority rule, or final decision by a senior expert after initial reads), but the exact "pre-defined" method (e.g., 2+1, 3+1) is not explicitly detailed beyond "up to three."
- Discrepant Analysis for Clinical Study: PCR testing followed by bi-directional sequencing was used for discrepant analysis between the device results and Giemsa microscopy. This serves as a molecular "tie-breaker" for cases where the human expert consensus diverges from the device.
Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study:
- No MRMC comparative effectiveness study was explicitly described in the provided text. The study primarily compares the device's performance to human expert microscopy as a reference standard (standalone performance relative to ground truth), rather than comparing human reader performance with and without AI assistance. The device is a "kit" for performing a FISH assay, observed by operators through a microscope, not an AI interpreting images for humans. So, an MRMC study in the typical AI sense is not applicable here.
Standalone Performance (Algorithm Only):
- Yes, the core of the clinical study evaluates the standalone performance of the ID-FISH Plasmodium Genus Test Kit (PlasG) and the ID-FISH Plasmodium falciparum and P. vivax Combo Test Kit (PlasFV). These are diagnostic kits based on FISH technology, not an automated image analysis algorithm in the AI sense. The "algorithm" here is the chemical reaction and visual interpretation by a human operator, making the performance measurement of the kit itself (its ability to bind to target RNA) rather than an AI algorithm's interpretive accuracy. The results presented in Tables 10-26 directly show the kit's performance (sensitivity, specificity, PPA, NPA) against the Giemsa microscopy gold standard, followed by PCR for discrepant cases.
Type of Ground Truth Used:
- Primary Ground Truth: Expert consensus Giemsa thick and thin smear malaria microscopy.
- Secondary/Discrepant Ground Truth: PCR testing followed by bi-directional sequencing for discrepant analysis between the device results and Giemsa microscopy. This acts as a higher-level confirmation for cases where the initial human reads and device results don't align.
Sample Size for Training Set:
- The document describes the analytical and clinical validation studies. There is no mention of a separate "training set" as would be typical for machine learning models. This is due to the nature of the device being a laboratory test kit (FISH assay) rather than an AI/ML diagnostic algorithm that requires a training phase. The studies described are for validation of the completed device.
How Ground Truth for Training Set was Established:
- As there's no explicitly mentioned "training set" in the context of an AI/ML model, this question is not directly applicable. If one considers the development of the FISH probes themselves, the "ground truth" would have been established through molecular biology techniques ensuring probe specificity and sensitivity to the target ribosomal RNA sequences. However, the document focuses on the validation of the final kit.

Summary

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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
1. Classification:
  Class II (Special Controls)

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3. Product code(s):

PYN

1. 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.

1. 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

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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

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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

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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%ConfidenceInterval
	Site 1		Site 2		Site 3
	Op 1	Op 2	Op 1	Op 2	Op 1	Op 2
Negative	30/30(100)	30/30(100)	30/30(100)	30/30(100)	30/30(100)	28a/29b(96.6)	178a/179b(99.4)	96.9 - 99.9%
Low P.f.	30/30(100)	30/30(100)	30/30(100)	30/30(100)	30/30(100)	28/28b(100)	178/178b(100)	97.9 – 100%
Mod. P.f.	30/30(100)	30/30(100)	30/30(100)	30/30(100)	30/30(100)	30/30(100)	180/180(100)	97.9-100%
Low P.v.	30/30(100)	30/30(100)	30/30(100)	30/30(100)	30/30(100)	30/30(100)	180/180(100)	97.9-100%
Mod. P.v.	30/30(100)	30/30(100)	30/30(100)	30/30(100)	30/30(100)	30/30(100)	180/180(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	ConfidenceInterval
Negative	30/30(100)	30/30(100)	30/30(100)	30/30(100)	30/30(100)	28a/29b(96.6)	178a/179b(99.4)	96.9 - 99.9%
Low P.f.	30/30(100)	30/30(100)	30/30(100)	30/30(100)	30/30(100)	27c/28b(96.4)	177c/178b(99.4)	96.9 - 99.9%
Mod. P.f.	30/30(100)	30/30(100)	30/30(100)	30/30(100)	30/30(100)	30/30(100)	180/180(100)	97.9 - 100%
Low P.v.	30/30(100)	30/30(100)	30/30(100)	30/30(100)	30/30(100)	30/30(100)	180/180(100)	97.9 - 100%
Mod. P.v	30/30(100)	30/30(100)	30/30(100)	30/30(100)	30/30(100)	30/30(100)	180/180(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

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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.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

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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.

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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

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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
1. Comparison Studies:
- a. Clinical Comparison:

See section L3

b. Matrix Equivalence Study
n/a
1. 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.

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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	TotalSamplesTested	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	TotalSamplesTested	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%

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Endemic/Non-Endemic	Country	Studyenrollment	#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).

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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).

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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
		P. f. Pos	P.f. Neg	Grand Total
PlasFV FISH	P.f. Pos	152	21a	173
	P.f. 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
		P.v. Pos	P.v. Neg	Grand Total
PlasFV FISH	P.v. Pos	156	9a	165
	P.v. 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.

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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
		P. f. Pos	P.f. Neg	Grand Total
PlasFV FISH	P.f. Pos	19	5a	24
	P.f. 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

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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
		P. f. Pos	P.f. Neg	Grand Total
PlasFV FISH	P.f. Pos	133	16a	149
	P.f. 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%

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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)

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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
		P. f. Pos	P.f. Neg	Grand Total
PlasFV FISH	P.f. Pos	5	0	5
	P.f. 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.

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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).

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Combined Results Stratified by Parasitemia - PlasG test kit

Parasites/µlblood	Sensitivity	(PlasG FISH Pos/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/µlblood	Sensitivity	(PlasG FISH Pos/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/μlblood	Sensitivity	(PlasG FISH Pos/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%

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Parasites/µlblood	Sensitivity	(PlasFV FISH Pos/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/ulblood	Sensitivity	(PlasFV FISH Pos/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.

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4. Clinical cut-off:

n/a. This is a qualitative test.

1. Expected values/Reference range:
  n/a

M. Instrument Name

n/a

N. System Descriptions:

1. Modes of Operation: n/a
1. Software:

n/a

1. Specimen Identification:
  n/a
1. Specimen Sampling and Handling:
  n/a
1. Calibration:
  n/a
1. 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.
1. 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.
1. Negative results limitation (Special Control 7)
  The device labeling must include a limitation that negative results do not preclude the possibility of infection.

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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 ofthe Benefit(s)	The PlasG and PlasFV FISH test kits are the first assays to use fluorescent in situhybridization to detect and identify malaria nucleic acids directly in blood smearsprepared from venous EDTA blood samples. The PlasG and PlasFV FISH test kits can assist microscopists with detecting andidentifying malaria parasites, and may be particularly valuable at sites where personnelhave less experience or training in interpreting malaria smears. The PlasG and PlasFV FISH test kits assist in distinguishing between Plasmodiumfalciparum and Plasmodium vivax infections. In comparison to expert adjudicated Giemsa microscopy, the PlasG and PlasFV FISHtest kits demonstrated sensitivity greater than 95% for the identification of Plasmodiumspp. and P. falciparum infections, and sensitivity greater than 90% for the identificationof P. vivax infections.
Summary ofthe Risk(s)	False positive results and false negative results are the primary risks associated withuse of the PlasG and PlasFV FISH test kits. A false positive result may lead to unnecessary antimalarial therapy, with associatedadverse events. A false negative result may result in a delay of antimalarial therapy, with subsequentworsening of infection. A false negative result may also result a missed opportunity toprovide antimalarial therapy to eliminate malaria hypnozoites, the dormant liver formof P. vivax, with subsequent relapse of infection.
Summary ofOtherFactors	None.

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Conclusions	The probable benefits of the PlasG and PlasFV FISH test kits outweigh the potential risks
Do theprobable	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
probablerisks?	likely to benefit patients by assisting with the diagnosis and identification of malariainfections. The high performance observed during the pivotal clinical trials in comparisonto adjudicated Giemsa microscopy, and the proposed special controls suggest that errorswill be uncommon and will be well mitigated by current laboratory practices, includingtraditional microscopy and product labelling. The PlasG and PlasFV FISH test kits couldprovide 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 clinicalspecimens
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.
- 1. Premarket notification submissions must include a detailed description of the fluorophores, signal source, detection mechanism and method of result interpretation.
- 1. 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.

1. 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.
1. 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.
1. 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.
1. The 21 CFR 809.10(b)(5)(ii) compliant labeling must include a limitation that negative results do not preclude the possibility of infection.

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Regulation Number and Section

§ 866.3367 Device to detect and identify microbial nucleic acids by FISH in clinical specimens.

(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). The special controls for this device are:(1) Design verification and validation must include the following:
(i) 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;
(ii) Detailed description of the fluorophores, signal source, detection mechanism, and method of result interpretation;
(iii) Detailed documentation from the following analytical studies: analytical sensitivity (Limit of Detection), inclusivity, reproducibility, interference, cross reactivity, and specimen stability; and
(iv) 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.
(2) The labeling required under § 809.10(b) of this chapter must include:
(i) A statement that the device is intended to be used in conjunction with clinical history, signs, symptoms, and the results of other diagnostic testing;
(ii) A detailed explanation of the interpretation of results and acceptance criteria for any quality control testing; and
(iii) A limitation that negative results do not preclude the possibility of infection.